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Cigarette smoke affects posttranslational modifications and inhibits capacitation-induced changes in human sperm proteins
Reproductive Toxicology 43 (2014) 125–129
Contents lists available at ScienceDirect
Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Cigarette smoke affects posttranslational modifications and inhibits capacitation-induced changes in human sperm proteins Vibha Shrivastava a , Hannah Marmor a , Sholom Chernyak a , Marc Goldstein b , Miriam Feliciano b , Margarita Vigodner a,c,∗ a
Department of Biology, Stern College, Yeshiva University, New York, NY, United States Department of Urology, Weill Cornell Medical College, New York, NY, United States c Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, United States b
a r t i c l e
i n f o
Article history: Received 3 July 2013 Received in revised form 27 November 2013 Accepted 5 December 2013 Available online 15 December 2013 Keywords: Sperm Tobacco Capacitation Sumoylation Phophorylation
a b s t r a c t Sperm are highly dependent on posttranslational modifications of proteins. Massive phosphorylation on tyrosine residue is required for sperm capacitation. Sumoylation has also been recently implicated in spermatogenesis and sperm functions. Cigarette smoke is known to cause oxidative stress in different tissues, and several studies suggest that it causes oxidative stress in sperm. Whether tobacco affects posttranslational modifications in human sperm is currently unknown. In this study, we show that a short exposure of human sperm to physiological concentrations of cigarette smoke extract (CSE) causes the partial de-sumoylation of many sperm proteins. Furthermore, the presence of a low concentration of CSE in the human tubal fluid during an induction of in vitro capacitation inhibits the capacitationassociated increase in protein phosphorylation. Collectively, changes in posttranslational modifications may be one of the mechanisms through which exposure to tobacco can negatively affect sperm functions and cause fertility problems. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Human sperm are produced in the testis through spermatogenesis, followed by their additional maturation in the epididymis. Before fertilization, sperm undergo a final activation in the female reproductive tract, known as capacitation, which involves the reorganization of the sperm plasma membranes and changes in the beating patterns of the flagella [1,2]. Capacitation is followed by an acrosome reaction and is a prerequisite for successful fertilization. At the molecular level, capacitation is hallmarked by the massive phosphorylation of sperm proteins on tyrosine residues; this process can be successfully induced and monitored in vitro. Beyond phosphorylation, various other posttranslational modifications have been detected in sperm, including nitrosilation, acetylation, and ubiquitination, but their role in sperm activity is less understood [3–5]. We have recently shown that numerous human sperm proteins are also modified by sumoylation (a
Abbreviations: HTF, human tubal fluid; CSE, cigarette smoke extract; HSA, human serum albumin. ∗ Corresponding author at: Department of Biology, Stern College, Yeshiva University, 245 Lexington Avenue, New York, NY 10016, United States. Tel.: +1 212 340 7769; fax: +1 212 340 7868. E-mail address: [email protected] (M. Vigodner). 0890-6238/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.reprotox.2013.12.001
covalent modification by small ubiquitin-like modifiers [SUMO proteins]). The identified targets of SUMO corresponded to flagella proteins, heat shock proteins, metabolic enzymes and the proteins involved in sperm maturation and differentiation [6]. Overall, a precisely regulated interplay between different posttranslational modifications should be very important for sperm because their transcription machinery is inactive. It has been demonstrated that cigarette smoke can adversely affect spermatogenesis and sperm functions. Sperm from smokers show higher level of DNA damage, apoptosis, and markers of oxidative stress (e.g., 8dg, lipid peroxidation) [7–11]. Tobacco severely affects sperm motility, the ability to undergo hyperactivation and acrosome reaction, bind zona pellucida and fertilize the egg, however the underlying mechanism is not well understood [7–17]. Studies in other tissues have shown that oxidative stress can affect the posttranslational modifications of proteins, such as sumoylation and phosphorylation; these changes in turn may play a role in stress-induced pathways [18]. We have recently shown that the in vivo exposure of mice to tobacco smoke can cause oxidation and de-sumoylation of proteins in testicular cells [19]. Whether tobacco affects posttranslational modifications in human sperm, including those required for sperm activation and capacitation, is not known. In this report, we show that a short exposure of human sperm to a physiological concentration of cigarette smoke extract (CSE) causes the partial de-sumoylation of sperm proteins. Furthermore,
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V. Shrivastava et al. / Reproductive Toxicology 43 (2014) 125–129
the presence of a low concentration of CSE in the human tubal during in vitro capacitation inhibits the capacitation-associated increase in protein phosphorylation. Collectively, changes in posttranslational modifications can be in part responsible for the negative effect of tobacco on sperm functions.
NaHCO3 . The sperm were pelleted and washed with 1 ml of HTF at 700 g for 10 min.
2. Materials and methods
For each condition, sperm pellets of approximately 3 × 106 sperm were resuspended in 80 L of 2× Laemmli buffer (126 mM of Tris/HCl, 20% Glycerol, 4% SDS) and boiled for 5 min at 100 ◦ C. After boiling, the samples were centrifuged for 2 min, and the supernatants were collected in fresh Eppendorf tubes. Protein concentrations were determined via a bicinchoninic acid protein assay using bovine serum albumin (BSA) as the standard (Pierce, Rockford, IL, USA). Before running the samples, betamercaptoethanol was added at 5% and Bromphenol blue at 0.02% of the sample volume, and the samples were boiled again for 3 min. Gel electrophoresis was performed under reducing conditions using NuPAGE 4–12% gradient Bis-Tris polyacrylamide gels and MOPS running buffer (Invitrogen, Carlsbad, CA, USA) at a constant 200 V. After electrophoresis, the proteins were transferred to a nitrocellulose membrane (0.45 m, Invitrogen, Carlsbad, CA, USA) using NuPAGE transfer buffer. Protein electrophoresis and transfer were performed with an Invitrogen XCell SureLock MiniCell electrophoresis system. Western blotting was performed using the ECL plus kit (GE Healthcare, Piscataway, NJ, USA), in accordance with the manufacturer’s instructions. SUMO1, SUMO2/3, and ubiquitin antibodies were used at 1:500 dilutions, and antiphosphotyrosine was used at a 1:1000 dilution in PBS containing 1% BSA and 0.1% sodium azide. Equal loading was ensured with a monoclonal anti--tubulin antibody (1:2000; Abcam, ab7291).
2.1. Reagents and antibodies Rabbit polyclonal antibodies against SUMO2/3 (ab3742) and ubiquitin (ab7780) were purchased from Abcam (Cambridge, MA, USA). The mouse G10 Platinum anti-phosphotyrosine antibody was obtained from Millipore (05-1050; Temecula, CA, USA). All remaining reagents were purchased from Sigma (St. Louis, MO, USA), unless otherwise noted. 2.2. Human sperm Sperm samples were obtained from the male fertility clinic at the Weill Cornell College of Medicine of Cornell University in New York, NY. Informed consent was obtained from all patients, in accordance with the protocol approved by the Institutional Review Board (IRB) of the Weill Cornell Medical College. Semen samples were obtained by masturbation after 3–5 days of sexual abstinence and were subjected to a routine seminal analysis of volume, sperm concentration, total sperm number per ejaculate, motility, vitality and normal morphology, according to the World Health Organization criteria (WHO, 1999). The human specimens received for the experiments did not contain any code derived from individual personal information. The samples were separated on 40–80% PureSperm gradient (Nidacon, Mölndal, Sweden), according to the manufacturer’s instructions. The fractions were pelleted and washed with 1 ml of human tubal fluid (HTF) medium (Irvine Scientific, Santa Ana, CA, USA) and centrifuged at 700 × g for 10 min. 2.3. Preparation of cigarette smoke extract and sperm treatment Cigarette smoke extract (CSE) was prepared as described previously [12,20–22]. In brief, one research cigarette (3R4F: 10 mg of tar and 0.8 mg of nicotine, Tobacco Research, University of Kentucky, Lexington, KY, USA) was attached to a tube connected to a Buchner flask that contained 25 mL PBS. The smoke derived from the cigarette was drawn into the flask under a vacuum generated by a nickel-plated water aspirator. The pH of the solution was then adjusted to 7.2–7.4 with 1 N HCl and filtered through a 0.22-m pore filter to remove bacteria and large particles. The resulting 100% CSE was diluted with PBS to achieve 1–20% concentrations and used within 30 min of preparation. In each treatment, approximately 3 × 106 cells were resuspended in 1.5 ml of PBS with or without CSE and incubated at 37 ◦ C with 5% CO2 for 1 h. Treatments were followed by the preparation of whole-cell protein lysates. Each experiment was repeated at least three times. 2.4. In vitro capacitation For some experiments, motile sperm fractions were used to induce in vitro capacitation. Sperm pellets consisting of approximately 3 × 106 cells were resuspended in 1.5 ml of HTF that was supplemented with human serum albumin (HSA, 5 mg/ml final concentration) and NaHCO3 (10 mM final concentration) and incubated with or without the addition of 5% CSE for 4 h at 37 ◦ C in 5% CO2 . The control sample was incubated without the addition of HSA and
2.5. Protein extraction and western blot analysis of SUMO expression
2.6. Densitometry and statistical analysis Quantitative densitometry analyses for SUMO, ubiquitin and phosphotyrosine were performed using the Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA) and the density values were normalized to tubulin. In each experiment, controls (untreated samples) were considered as 100% and other samples were normalized to the controls. The results were expressed as the means ± standard deviation (SD). To calculate the difference between samples, Student’s paired t-test was used. p values <0.05 were considered statistically significant. 3. Results and discussion To gain insights into possible effects of CSE exposure on sumoylation in sperm, western blot analyses were performed to compare the levels of high-molecular weight SUMO conjugates in cells subjected to different concentrations of CSE. The concentrations of 1–5% CSE were chosen because the nicotine concentrations in this extract were similar to the blood concentrations measured in the blood of the smokers [12,21]. The 1–5% CSE range was used in several previously published studies [20,22]. Despite some slight variations among different sperm samples in their response to CSE, a decrease in sumoylation of most high molecular weight proteins was significant in the range of 1–5% CSE (p < 0.05, n = 5; Fig. 1A, two different samples are shown). The exception was prominent bands at around 90 kDa which showed either no change or an increase in the intensity (Fig. 1A, arrows); those bands were excluded from the densitometry analysis. Thus, similar to the results obtained with mouse germ cells in vivo, a short exposure of human sperm to CSE caused de-sumoylation of many proteins, even at concentrations as low as 1%. It has been shown in other tissues that oxidative
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Fig. 1. Effect of cigarette smoke extract (CSE) on protein sumoylation in human sperm. Sperm were incubated for one hour with increasing concentrations of freshly prepared CSE (1–5% in PBS), followed by the preparation of whole-cell protein lysates. Western blot analysis was performed using anti-SUMO2/3 (A) or anti-ubiquitin (B) antibodies. Equal loading was monitored using a monoclonal anti--tubulin antibody. M, molecular weight markers; positions of the molecular weight markers are indicated. Samples from two different normal donors are shown in (A). Bands at around 90 kDa which showed either no change or an increase in the intensity (A, arrows) were excluded from the densitometry analysis. Density values for SUMO and ubiquitin were normalized to tubulin. In each experiment, controls (untreated samples) were considered as 100% and other samples were normalized to the controls. The results were expressed as the means ± standard deviation (SD). Significant reductions in protein sumoylation (p values <0.05, n = 5), but no changes in the levels of ubiquitinated proteins, were detected.
stress at physiological (low) concentrations can inactivate SUMOactivating and conjugating enzymes through the formation of a disulfide bond between these proteins, thus causing partial protein de-sumoylation [19,23]. Our published results in testicular cells [19] and the data obtained in this study are consistent with these findings, as a statistically significant decrease in sumoylation was observed in the range of 1–5% CSE. At higher doses of oxidative stress, both the conjugating and the de-conjugating machinery can be inactivated and cell death is often initiated [19,23]; in line with these findings, either no changes or an increase in the level of sumoylated proteins were observed in sperm samples treated with high concentrations of CSE (10 and 20%). Interestingly, the decrease in sumoylation at the range of 1–5% was not related to a proteasome activity because the levels of ubiquitinated proteins treated with the same CSE concentrations did not change (Fig. 1B). Our group has recently identified numerous sumoylated proteins implicated in sperm motility and metabolism; the desumoylation of these proteins can modify their activity and negatively affect sperm functions. Interestingly, abnormally high sumoylation is also associated with decreased sperm quality [6]. It is possible that the level of protein sumoylation in sperm should be maintained in a relatively narrow normal range. Further studies are needed for
confirming the effect of CSE on the sperm sumoylation machinery and for uncovering the functional consequences of desumoylation of specific targets. Sperm capacitation is hallmarked by massive phosphorylation on tyrosine resides. To gain insights into effects of CSE on capacitation, the levels of phosphotyrosine were compared in control samples and samples that underwent capacitation with or without the presence of 5% CSE. The successful progression of capacitation was monitored using anti-phosphotyrosine antibodies in western blot and fluorescent microscopy analyses (Fig. 2A and B). Our western blot results show that the presence of CSE in the human tubal fluid (HTF) severely inhibited the increase in protein phosphorylation upon the induction of capacitation (p < 0.05, n = 5; Fig. 2A, two different samples are shown). The tyrosine phosphorylation of several flagella proteins was shown to be directly related to the successful acquisition of sperm hyperactive motility and to the penetration of the cumulus and the zona pellucida of the oocyte [24,25]. The major targets of phosphorylation are several protein A-kinase anchoring proteins (AKAPs) localized on the fibrous sheath and regulating sperm motility [26,27]. Importantly, mice lacking the AKAP4 protein are infertile, as their sperm fails to show progressive motility [28].
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Fig. 2. Effect of cigarette smoke extract (CSE) on sperm capacitation. To induce capacitation (cap), sperm were resuspended in HTF supplemented with human serum albumin and NaHCO3 and incubated with or without the addition of 5% cigarette smoke extract (CSE) for 4 h, as described in Section 2. The control sample (C) was incubated without the addition of HSA and NaHCO3 . Western blot analysis was performed using anti-phosphotyrosine antibody. Samples from two different normal donors are shown (Fig. 2A). Equal loading was monitored using a monoclonal anti--tubulin antibody. Positions of molecular weight markers are indicated. Density values for phospotyrosine were normalized to tubulin. In each experiment, controls (untreated samples) were considered as 100% and other samples were normalized to the controls. The results were expressed as the means ± standard deviation (SD). Capacitation was associated with a significant increase in phophorylation on tyrosine residues (p < 0.05, n = 5; Fig. 2A). The presence of CSE in the HTF severely inhibited the increase in protein phosphorylation (p < 0.05, n = 5; Fig. 2A). The successful progression of capacitation was also monitored microscopically (B; sperm tailes are immunostained using anti-phosphotyrosine antibody.
The phosphorylation of several glycolytic enzymes also appears to be important for energy metabolism during capacitation [29]. Our data provide evidence for the first time that tobacco severely affects capacitation-associated phophorylation events in sperm. These changes can be responsible for CSE-induced decreases in sperm motility and activation upon exposure to tobacco. Interestingly, AKAPs have also been identified by our group as targets of sumoylation; the interplay between sumoylation and phosphorylation would thus be interesting to study further.
Transparency documents associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.reprotox.2013.12.001. Funding This study was supported by a grant from the Flight Attendant Medical Research Institute (to MV). References
4. Conclusions Altogether, our data suggest that tobacco causes changes in the sumoylation and inhibits phosphorylation of sperm proteins required for capacitation. While sperm maturation occurs in males, capacitation occurs in females. Therefore, this study uncovers an additional mechanism through which the exposure of both men and women to tobacco can potentially lead to infertility, impaired fertilization or birth defects. Conflicts of interest The authors have no conflicts of interest to declare.
[1] Visconti PE. Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. Development 1995;121(4):1129–37. [2] Visconti PE. Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Development 1995;121(4):1139–50. [3] Lefievre L. Human spermatozoa contain multiple targets for protein S-nitrosylation: an alternative mechanism of the modulation of sperm function by nitric oxide? Proteomics 2007;7(17):3066–84. [4] Sutovsky P. Ubiquitin-dependent proteolysis in mammalian spermatogenesis, fertilization, and sperm quality control: killing three birds with one stone. Microsc Res Tech 2003;61(1):88–102. [5] Sutovsky P, Terada Y, Schatten G. Ubiquitin-based sperm assay for the diagnosis of male factor infertility. Hum Reprod 2001;16(2):250–8. [6] Vigodner M. Localization and identification of sumoylated proteins in human sperm: excessive sumoylation is a marker of defective spermatozoa. Hum Reprod 2013;28(1):210–23.
V. Shrivastava et al. / Reproductive Toxicology 43 (2014) 125–129 [7] Fariello RM. Effect of smoking on the functional aspects of sperm and seminal plasma protein profiles in patients with varicocele. Hum Reprod 2012;27(11):3140–9. [8] Kiziler AR. High levels of cadmium and lead in seminal fluid and blood of smoking men are associated with high oxidative stress and damage in infertile subjects. Biol Trace Elem Res 2007;120(1–3):82–91. [9] Laudat A, Lecourbe K, Palluel AM. Tobacco: a potential inductor of lipid peroxidation of the human spermatozoon membrane? Ann Biol Clin (Paris) 2004;62(6):681–6. [10] DeMarini DM. Genotoxicity of tobacco smoke and tobacco smoke condensate: a review. Mutat Res 2004;567(2–3):447–74. [11] Ji G. OGG1 Ser326Cys polymorphism interacts with cigarette smoking to increase oxidative DNA damage in human sperm and the risk of male infertility. Toxicol Lett 2013;218(2):144–9. [12] Calogero A. Cigarette smoke extract immobilizes human spermatozoa and induces sperm apoptosis. Reprod Biomed Online 2009;19(4): 564–71. [13] Marczylo EL. Smoking induces differential miRNA expression in human spermatozoa: a potential transgenerational epigenetic concern? Epigenetics 2012;7(5):432–9. [14] Kulikauskas V, Blaustein D, Ablin RJ. Cigarette smoking and its possible effects on sperm. Fertil Steril 1985;44(4):526–8. [15] Hung PH. Effects of environmental tobacco smoke in vitro on rhesus monkey sperm function. Reprod Toxicol 2007;23(4):499–506. [16] Sofikitis N. Effects of cotinine on sperm motility: membrane function, and fertilizing capacity in vitro. Urol Res 2000;28(6):370–5. [17] Talbot P, Lin S. The effect of cigarette smoke on fertilization and pre-implantation development: assessment using animal models, clinical data, and stem cells. Biol Res 2011;44(2):189–94. [18] Tempe D, Piechaczyk M, Bossis G. SUMO under stress. Biochem Soc Trans 2008;36(Pt 5):874–8.
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[19] Shrivastava V. SUMO proteins are involved in the stress response during spermatogenesis and are localized to DNA double-strand breaks in germ cells. Reproduction 2010;139(6):999–1010. [20] Mercer BA. Identification of a cigarette smoke-responsive region in the distal MMP-1 promoter. Am J Respir Cell Mol Biol 2009;40(1):4–12. [21] Lawson GM. Application of serum nicotine and plasma cotinine concentrations to assessment of nicotine replacement in light: moderate, and heavy smokers undergoing transdermal therapy. J Clin Pharmacol 1998;38(6):502–9. [22] Lemaitre V, Dabo AJ, D’Armiento J. Cigarette smoke components induce matrix metalloproteinase-1 in aortic endothelial cells through inhibition of mTOR signaling. Toxicol Sci 2011;123(2):542–9. [23] Bossis G, Melchior F. Regulation of SUMOylation by reversible oxidation of SUMO conjugating enzymes. Mol Cell 2006;21(3):p349–57. [24] Nassar A. Modulation of sperm tail protein tyrosine phosphorylation by pentoxifylline and its correlation with hyperactivated motility. Fertil Steril 1999;71(5):919–23. [25] Naz RK, Rajesh PB. Role of tyrosine phosphorylation in sperm capacitation/acrosome reaction. Reprod Biol Endocrinol 2004;2:p75. [26] Carrera A. Regulation of protein tyrosine phosphorylation in human sperm by a calcium/calmodulin-dependent mechanism: identification of A kinase anchor proteins as major substrates for tyrosine phosphorylation. Dev Biol 1996;180(1):284–96. [27] Mandal A. FSP95: a testis-specific 95-kilodalton fibrous sheath antigen that undergoes tyrosine phosphorylation in capacitated human spermatozoa. Biol Reprod 1999;61(5):1184–97. [28] Miki K. Targeted disruption of the Akap4 gene causes defects in sperm flagellum and motility. Dev Biol 2002;248(2):331–42. [29] Ficarro S. Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation. J Biol Chem 2003;278(13):11579–89.
Contents lists available at ScienceDirect
Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Cigarette smoke affects posttranslational modifications and inhibits capacitation-induced changes in human sperm proteins Vibha Shrivastava a , Hannah Marmor a , Sholom Chernyak a , Marc Goldstein b , Miriam Feliciano b , Margarita Vigodner a,c,∗ a
Department of Biology, Stern College, Yeshiva University, New York, NY, United States Department of Urology, Weill Cornell Medical College, New York, NY, United States c Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, United States b
a r t i c l e
i n f o
Article history: Received 3 July 2013 Received in revised form 27 November 2013 Accepted 5 December 2013 Available online 15 December 2013 Keywords: Sperm Tobacco Capacitation Sumoylation Phophorylation
a b s t r a c t Sperm are highly dependent on posttranslational modifications of proteins. Massive phosphorylation on tyrosine residue is required for sperm capacitation. Sumoylation has also been recently implicated in spermatogenesis and sperm functions. Cigarette smoke is known to cause oxidative stress in different tissues, and several studies suggest that it causes oxidative stress in sperm. Whether tobacco affects posttranslational modifications in human sperm is currently unknown. In this study, we show that a short exposure of human sperm to physiological concentrations of cigarette smoke extract (CSE) causes the partial de-sumoylation of many sperm proteins. Furthermore, the presence of a low concentration of CSE in the human tubal fluid during an induction of in vitro capacitation inhibits the capacitationassociated increase in protein phosphorylation. Collectively, changes in posttranslational modifications may be one of the mechanisms through which exposure to tobacco can negatively affect sperm functions and cause fertility problems. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Human sperm are produced in the testis through spermatogenesis, followed by their additional maturation in the epididymis. Before fertilization, sperm undergo a final activation in the female reproductive tract, known as capacitation, which involves the reorganization of the sperm plasma membranes and changes in the beating patterns of the flagella [1,2]. Capacitation is followed by an acrosome reaction and is a prerequisite for successful fertilization. At the molecular level, capacitation is hallmarked by the massive phosphorylation of sperm proteins on tyrosine residues; this process can be successfully induced and monitored in vitro. Beyond phosphorylation, various other posttranslational modifications have been detected in sperm, including nitrosilation, acetylation, and ubiquitination, but their role in sperm activity is less understood [3–5]. We have recently shown that numerous human sperm proteins are also modified by sumoylation (a
Abbreviations: HTF, human tubal fluid; CSE, cigarette smoke extract; HSA, human serum albumin. ∗ Corresponding author at: Department of Biology, Stern College, Yeshiva University, 245 Lexington Avenue, New York, NY 10016, United States. Tel.: +1 212 340 7769; fax: +1 212 340 7868. E-mail address: [email protected] (M. Vigodner). 0890-6238/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.reprotox.2013.12.001
covalent modification by small ubiquitin-like modifiers [SUMO proteins]). The identified targets of SUMO corresponded to flagella proteins, heat shock proteins, metabolic enzymes and the proteins involved in sperm maturation and differentiation [6]. Overall, a precisely regulated interplay between different posttranslational modifications should be very important for sperm because their transcription machinery is inactive. It has been demonstrated that cigarette smoke can adversely affect spermatogenesis and sperm functions. Sperm from smokers show higher level of DNA damage, apoptosis, and markers of oxidative stress (e.g., 8dg, lipid peroxidation) [7–11]. Tobacco severely affects sperm motility, the ability to undergo hyperactivation and acrosome reaction, bind zona pellucida and fertilize the egg, however the underlying mechanism is not well understood [7–17]. Studies in other tissues have shown that oxidative stress can affect the posttranslational modifications of proteins, such as sumoylation and phosphorylation; these changes in turn may play a role in stress-induced pathways [18]. We have recently shown that the in vivo exposure of mice to tobacco smoke can cause oxidation and de-sumoylation of proteins in testicular cells [19]. Whether tobacco affects posttranslational modifications in human sperm, including those required for sperm activation and capacitation, is not known. In this report, we show that a short exposure of human sperm to a physiological concentration of cigarette smoke extract (CSE) causes the partial de-sumoylation of sperm proteins. Furthermore,
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V. Shrivastava et al. / Reproductive Toxicology 43 (2014) 125–129
the presence of a low concentration of CSE in the human tubal during in vitro capacitation inhibits the capacitation-associated increase in protein phosphorylation. Collectively, changes in posttranslational modifications can be in part responsible for the negative effect of tobacco on sperm functions.
NaHCO3 . The sperm were pelleted and washed with 1 ml of HTF at 700 g for 10 min.
2. Materials and methods
For each condition, sperm pellets of approximately 3 × 106 sperm were resuspended in 80 L of 2× Laemmli buffer (126 mM of Tris/HCl, 20% Glycerol, 4% SDS) and boiled for 5 min at 100 ◦ C. After boiling, the samples were centrifuged for 2 min, and the supernatants were collected in fresh Eppendorf tubes. Protein concentrations were determined via a bicinchoninic acid protein assay using bovine serum albumin (BSA) as the standard (Pierce, Rockford, IL, USA). Before running the samples, betamercaptoethanol was added at 5% and Bromphenol blue at 0.02% of the sample volume, and the samples were boiled again for 3 min. Gel electrophoresis was performed under reducing conditions using NuPAGE 4–12% gradient Bis-Tris polyacrylamide gels and MOPS running buffer (Invitrogen, Carlsbad, CA, USA) at a constant 200 V. After electrophoresis, the proteins were transferred to a nitrocellulose membrane (0.45 m, Invitrogen, Carlsbad, CA, USA) using NuPAGE transfer buffer. Protein electrophoresis and transfer were performed with an Invitrogen XCell SureLock MiniCell electrophoresis system. Western blotting was performed using the ECL plus kit (GE Healthcare, Piscataway, NJ, USA), in accordance with the manufacturer’s instructions. SUMO1, SUMO2/3, and ubiquitin antibodies were used at 1:500 dilutions, and antiphosphotyrosine was used at a 1:1000 dilution in PBS containing 1% BSA and 0.1% sodium azide. Equal loading was ensured with a monoclonal anti--tubulin antibody (1:2000; Abcam, ab7291).
2.1. Reagents and antibodies Rabbit polyclonal antibodies against SUMO2/3 (ab3742) and ubiquitin (ab7780) were purchased from Abcam (Cambridge, MA, USA). The mouse G10 Platinum anti-phosphotyrosine antibody was obtained from Millipore (05-1050; Temecula, CA, USA). All remaining reagents were purchased from Sigma (St. Louis, MO, USA), unless otherwise noted. 2.2. Human sperm Sperm samples were obtained from the male fertility clinic at the Weill Cornell College of Medicine of Cornell University in New York, NY. Informed consent was obtained from all patients, in accordance with the protocol approved by the Institutional Review Board (IRB) of the Weill Cornell Medical College. Semen samples were obtained by masturbation after 3–5 days of sexual abstinence and were subjected to a routine seminal analysis of volume, sperm concentration, total sperm number per ejaculate, motility, vitality and normal morphology, according to the World Health Organization criteria (WHO, 1999). The human specimens received for the experiments did not contain any code derived from individual personal information. The samples were separated on 40–80% PureSperm gradient (Nidacon, Mölndal, Sweden), according to the manufacturer’s instructions. The fractions were pelleted and washed with 1 ml of human tubal fluid (HTF) medium (Irvine Scientific, Santa Ana, CA, USA) and centrifuged at 700 × g for 10 min. 2.3. Preparation of cigarette smoke extract and sperm treatment Cigarette smoke extract (CSE) was prepared as described previously [12,20–22]. In brief, one research cigarette (3R4F: 10 mg of tar and 0.8 mg of nicotine, Tobacco Research, University of Kentucky, Lexington, KY, USA) was attached to a tube connected to a Buchner flask that contained 25 mL PBS. The smoke derived from the cigarette was drawn into the flask under a vacuum generated by a nickel-plated water aspirator. The pH of the solution was then adjusted to 7.2–7.4 with 1 N HCl and filtered through a 0.22-m pore filter to remove bacteria and large particles. The resulting 100% CSE was diluted with PBS to achieve 1–20% concentrations and used within 30 min of preparation. In each treatment, approximately 3 × 106 cells were resuspended in 1.5 ml of PBS with or without CSE and incubated at 37 ◦ C with 5% CO2 for 1 h. Treatments were followed by the preparation of whole-cell protein lysates. Each experiment was repeated at least three times. 2.4. In vitro capacitation For some experiments, motile sperm fractions were used to induce in vitro capacitation. Sperm pellets consisting of approximately 3 × 106 cells were resuspended in 1.5 ml of HTF that was supplemented with human serum albumin (HSA, 5 mg/ml final concentration) and NaHCO3 (10 mM final concentration) and incubated with or without the addition of 5% CSE for 4 h at 37 ◦ C in 5% CO2 . The control sample was incubated without the addition of HSA and
2.5. Protein extraction and western blot analysis of SUMO expression
2.6. Densitometry and statistical analysis Quantitative densitometry analyses for SUMO, ubiquitin and phosphotyrosine were performed using the Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA) and the density values were normalized to tubulin. In each experiment, controls (untreated samples) were considered as 100% and other samples were normalized to the controls. The results were expressed as the means ± standard deviation (SD). To calculate the difference between samples, Student’s paired t-test was used. p values <0.05 were considered statistically significant. 3. Results and discussion To gain insights into possible effects of CSE exposure on sumoylation in sperm, western blot analyses were performed to compare the levels of high-molecular weight SUMO conjugates in cells subjected to different concentrations of CSE. The concentrations of 1–5% CSE were chosen because the nicotine concentrations in this extract were similar to the blood concentrations measured in the blood of the smokers [12,21]. The 1–5% CSE range was used in several previously published studies [20,22]. Despite some slight variations among different sperm samples in their response to CSE, a decrease in sumoylation of most high molecular weight proteins was significant in the range of 1–5% CSE (p < 0.05, n = 5; Fig. 1A, two different samples are shown). The exception was prominent bands at around 90 kDa which showed either no change or an increase in the intensity (Fig. 1A, arrows); those bands were excluded from the densitometry analysis. Thus, similar to the results obtained with mouse germ cells in vivo, a short exposure of human sperm to CSE caused de-sumoylation of many proteins, even at concentrations as low as 1%. It has been shown in other tissues that oxidative
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Fig. 1. Effect of cigarette smoke extract (CSE) on protein sumoylation in human sperm. Sperm were incubated for one hour with increasing concentrations of freshly prepared CSE (1–5% in PBS), followed by the preparation of whole-cell protein lysates. Western blot analysis was performed using anti-SUMO2/3 (A) or anti-ubiquitin (B) antibodies. Equal loading was monitored using a monoclonal anti--tubulin antibody. M, molecular weight markers; positions of the molecular weight markers are indicated. Samples from two different normal donors are shown in (A). Bands at around 90 kDa which showed either no change or an increase in the intensity (A, arrows) were excluded from the densitometry analysis. Density values for SUMO and ubiquitin were normalized to tubulin. In each experiment, controls (untreated samples) were considered as 100% and other samples were normalized to the controls. The results were expressed as the means ± standard deviation (SD). Significant reductions in protein sumoylation (p values <0.05, n = 5), but no changes in the levels of ubiquitinated proteins, were detected.
stress at physiological (low) concentrations can inactivate SUMOactivating and conjugating enzymes through the formation of a disulfide bond between these proteins, thus causing partial protein de-sumoylation [19,23]. Our published results in testicular cells [19] and the data obtained in this study are consistent with these findings, as a statistically significant decrease in sumoylation was observed in the range of 1–5% CSE. At higher doses of oxidative stress, both the conjugating and the de-conjugating machinery can be inactivated and cell death is often initiated [19,23]; in line with these findings, either no changes or an increase in the level of sumoylated proteins were observed in sperm samples treated with high concentrations of CSE (10 and 20%). Interestingly, the decrease in sumoylation at the range of 1–5% was not related to a proteasome activity because the levels of ubiquitinated proteins treated with the same CSE concentrations did not change (Fig. 1B). Our group has recently identified numerous sumoylated proteins implicated in sperm motility and metabolism; the desumoylation of these proteins can modify their activity and negatively affect sperm functions. Interestingly, abnormally high sumoylation is also associated with decreased sperm quality [6]. It is possible that the level of protein sumoylation in sperm should be maintained in a relatively narrow normal range. Further studies are needed for
confirming the effect of CSE on the sperm sumoylation machinery and for uncovering the functional consequences of desumoylation of specific targets. Sperm capacitation is hallmarked by massive phosphorylation on tyrosine resides. To gain insights into effects of CSE on capacitation, the levels of phosphotyrosine were compared in control samples and samples that underwent capacitation with or without the presence of 5% CSE. The successful progression of capacitation was monitored using anti-phosphotyrosine antibodies in western blot and fluorescent microscopy analyses (Fig. 2A and B). Our western blot results show that the presence of CSE in the human tubal fluid (HTF) severely inhibited the increase in protein phosphorylation upon the induction of capacitation (p < 0.05, n = 5; Fig. 2A, two different samples are shown). The tyrosine phosphorylation of several flagella proteins was shown to be directly related to the successful acquisition of sperm hyperactive motility and to the penetration of the cumulus and the zona pellucida of the oocyte [24,25]. The major targets of phosphorylation are several protein A-kinase anchoring proteins (AKAPs) localized on the fibrous sheath and regulating sperm motility [26,27]. Importantly, mice lacking the AKAP4 protein are infertile, as their sperm fails to show progressive motility [28].
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Fig. 2. Effect of cigarette smoke extract (CSE) on sperm capacitation. To induce capacitation (cap), sperm were resuspended in HTF supplemented with human serum albumin and NaHCO3 and incubated with or without the addition of 5% cigarette smoke extract (CSE) for 4 h, as described in Section 2. The control sample (C) was incubated without the addition of HSA and NaHCO3 . Western blot analysis was performed using anti-phosphotyrosine antibody. Samples from two different normal donors are shown (Fig. 2A). Equal loading was monitored using a monoclonal anti--tubulin antibody. Positions of molecular weight markers are indicated. Density values for phospotyrosine were normalized to tubulin. In each experiment, controls (untreated samples) were considered as 100% and other samples were normalized to the controls. The results were expressed as the means ± standard deviation (SD). Capacitation was associated with a significant increase in phophorylation on tyrosine residues (p < 0.05, n = 5; Fig. 2A). The presence of CSE in the HTF severely inhibited the increase in protein phosphorylation (p < 0.05, n = 5; Fig. 2A). The successful progression of capacitation was also monitored microscopically (B; sperm tailes are immunostained using anti-phosphotyrosine antibody.
The phosphorylation of several glycolytic enzymes also appears to be important for energy metabolism during capacitation [29]. Our data provide evidence for the first time that tobacco severely affects capacitation-associated phophorylation events in sperm. These changes can be responsible for CSE-induced decreases in sperm motility and activation upon exposure to tobacco. Interestingly, AKAPs have also been identified by our group as targets of sumoylation; the interplay between sumoylation and phosphorylation would thus be interesting to study further.
Transparency documents associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.reprotox.2013.12.001. Funding This study was supported by a grant from the Flight Attendant Medical Research Institute (to MV). References
4. Conclusions Altogether, our data suggest that tobacco causes changes in the sumoylation and inhibits phosphorylation of sperm proteins required for capacitation. While sperm maturation occurs in males, capacitation occurs in females. Therefore, this study uncovers an additional mechanism through which the exposure of both men and women to tobacco can potentially lead to infertility, impaired fertilization or birth defects. Conflicts of interest The authors have no conflicts of interest to declare.
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