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Anti-oxidative and anti-apoptotic roles of spermatogonial stem cells in reversing cisplatin-induced testicular toxicity
Cytotherapy, 2015; 0: 1e9
Anti-oxidative and anti-apoptotic roles of spermatogonial stem cells in reversing cisplatin-induced testicular toxicity
YOUSRI M. HUSSEIN1, RANDA H. MOHAMED2, SALLY M. SHALABY2, MANAL R. ABD EL-HALEEM3 & DALIA M. ABD EL MOTTELEB4 1
Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt, Medical Laboratory Department, Faculty of Applied Medical Science, Taif University, Taif, KSA, 2Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt, 3Histology and Cell Biology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt, Histology Department, Faculty of Oral Medicine and Dentistry, MTI University, Cairo, Egypt and 4Clinical Pharmacology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt. Abstract Background aims. Because of reproductive toxic effects of chemotherapy, researchers have taken some techniques to preserve fertility potential. The present study was designed to point out the potential role of spermatogonial stem cell (SSC) therapy in reversing cisplatin (CP)-induced testicular toxicity and restore the spermatogenesis. Methods. Sixty rats were randomly divided into three groups: group 1, control group; group 2, rats received CP in a dose of 7 mg/kg/day for 5 consecutive days; group 3, CP was injected at 7 mg/kg per day for 5 consecutive days, and, on the 6th day of the experiment, rats were treated with SSC. Forty days after receiving the last dose of CP, rats were euthanized under anesthesia; testes were collected, and gene expression using real-time polymerase chain reaction for P53, Bax, caspase 9 and cytochrome c, testicular histological findings and oxidative status were determined. Results. Administration of cisplatin caused significant increases in malondialdehyde levels, Bax and caspase 9 genes expression levels concomitant with significant decreases in anti-oxidant enzyme activities, p53 and cytochrome c gene expression levels, along with some histopathological lesions in testicular tissue. SCC attenuated the disturbance in oxidant/anti-oxidant status and testicular apoptosis; this is associated with improvements in the histopathological view of the testicular tissue. Conclusions. The current study highlights evidence that the SCC has antioxidative and anti-apoptotic properties that could reverse CP-induced testicular toxicity, in addition to their role in spermatogenesis. Key Words: anti-apoptotic, anti-oxidant, cisplatin, infertility, spermatogonial stem cell
Introduction Cisplatin (CP-diamminedichloroplatinum-II, CP) is a highly effective chemotherapeutic agent used for the treatment of many types of cancers including sarcomas, lymphomas, testicular, ovarian, breast, lung and bladder cancer [1,2]. Despite the broadspectrum clinical applications of CP, severe toxic side effects such as reproductive toxicity and increased the frequency of germ cell apoptosis, constitute a major consequence to its use [3,4]. The precise mechanism by which CP causes testicular toxicity and germ cell apoptosis is not fully known; however, numerous studies have shown that CP exposure can disrupt the redox balance of tissues, suggesting that biochemical and physiological disturbances may result from oxidative stress [5,6].
Reactive oxygen species are normally generated in mitochondria of testes, which are subsequently scavenged by anti-oxidant defense systems of the corresponding cellular compartments [7]. However, this balance can easily be broken by chemicals such as cisplatin, which disrupt the pro-oxidanteantioxidant balance, leading to cellular dysfunction [6]. Researchers have used some techniques such as the development of a spermatogonial transplantation, which provided a new treatment strategy for male infertility after the use of chemotherapy and irradiation [8]. The spermatogonial stem cells (SSC) colonize and initiate spermatogenesis, after the transplantation of dissociated testis cells into a seminiferous tubule microenvironment [9]. Because SSC self-renew and differentiate into proliferating spermatogonia, they
Correspondence: Randa H. Mohamed, MD, Faculty of Medicine, Zagazig University, 44519 Zagazig, Egypt. E-mail: [email protected] (Received 29 January 2015; accepted 5 July 2015) ISSN 1465-3249 Copyright Ó 2015, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2015.07.001
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provide a limitless supply of mature spermatozoa. Thus, SSC transplantation may be useful for the treatment of male infertility. It may also offer a great help in cancer patients who are undergoing stem celldestroying irradiation or chemotherapy, by prior isolation of stem cells and autotransplantation [10]. The purpose of the present study was to assess the potential role of stem cell therapy in reversing cisplatin-induced testicular toxicity and on antioxidant and anti-apoptotic capacity. Methods Chemicals Cisplatin (10 mg/10 mL) was from Sigma-Aldrich Inc. The other chemicals were obtained from Sigma. Preparation of donor cells and transplantation Donor testes were prepared according to Nayernia et al. [11]. Testes were isolated from four or five white albino rats (age between 5e10 days), washed in phosphate-buffered saline, and put in 5 mL of collagenase/Hanks balanced salt solution (pH 7.4) in a 60-mm sterile Petri dish. It was then incubated for 10 to 15 min at 37 C with gentle agitation on a rocker platform until the tubules separated. The tubules were poured off and transferred to a 10-mL cell culture centrifugation tube and centrifuged at 4 C at 650g for 5 min. The supernatant was added to 5 mL of Hanks’ balanced salt solution and centrifuged again at 4 C at 650g for 5 min. Dispase solution (2 mL) was added and stirred slowly at 37 C until the tissue was sufficiently dissolved (30 min). Dispersion of the tubule cells can be hastened by pipetting and gentle agitation. Fresh dispase solution was added to the remaining tubule fragment if further disaggregation is required. Cells were pelleted by centrifugation at 16 C at 650g for 5 min. The pellet was resuspended in 400 mL of Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). The cells can be maintained for 1 to 5 h at 4 C until transplantation. Trypan blue (0.3%) was added, and the cells were transplanted into the seminiferous tubules through the efferent ductules of recipient rat.
ad libitum access to standard rodent chow diet and filtered water. The rats were allowed to acclimate for 1 week before any use in experiments. All experimental procedures were approved by the local authorities, that is, the Ethical Committee for Animal Handling at Zagazig University, and were conducted according to the guidelines set forth by the National Institutes of Health (USA). For the experiment, rats were divided into three groups containing 20 rats per group and received the following: the control group received 10 mL of DMEM containing 10% FBS and trypan blue (0.3%) (vehicle of SSC) through injection into the testes for 6 consecutive days of the experiment. Group 2 rats were treated with CP alone: CP was injected (intra-peritoneally) in a dose of 7 mg/kg per day for 5 consecutive days. On the 6th day of the experiment, injections of 10 mL of DMEM containing 10% FBS and trypan blue (0.3%) (vehicle of SSC) to the testes via inserting a needle through the efferent ductules outside of the testis and passing the needle into the rete testis. In group 3, CP was injected intra-peritoneally at 7 mg/kg per day for 5 consecutive days. On the 6th day of experiment, rats received 1 106 cells SSC dissolved in 10 mL of DMEM containing 10% FBS and trypan blue (0.3%) in a single dose via inserting a needle through the efferent ductules outside of the testis and passing the needle into the rete testis. The testicular toxicity of CP can be detected within 7 weeks after treatment with a sufficient dose in rats [12]. The rats were killed under anesthesia (ketamine, 200 mg/kg intraperitoneally) at the end of the treatment period. Biochemical analysis For determination of lipid peroxidation levels, testicular tissue lipid peroxide levels (as malondialdehyde, MDA) were measured with the use of the thiobarbituric acid (TBA) reaction method of Ohkawa et al. [13]. The testis was decapsulated and homogenized in 10% trichloroacetic acid. MDA reacted with freshly prepared TBA to form a colored complex, and the absorption of the supernatant was recorded at 532 nm by comparing the absorption with the standard curve of MDA equivalents generated by acid-catalyzed hydrolysis of 1,1,3,3tetramethoxypropane.
Animals and experimental design Male Wistar rats (n ¼ 60; 8e10 weeks old; 200e220 g) were obtained from the Faculty of Veterinary Medicine at Zagazig University. On arrival, rats were housed under constant environmental conditions (room temperature 25 2 C with a 12-h light/dark cycle and 50% humidity). Rats were provided
Anti-oxidant enzyme assay Catalase activity (CAT) was determined according to the method of Aebi [14]. The testis was homogenized at 4 C in potassium phosphate buffer (50 mmol/L potassium phosphate and 1 mmol/L ethylenediamine tetra-acetic acid, pH 7) and centrifuged at 3500 rpm
Spermatogonial stem cells Table I. Sequence of the primers used for real-time polymerase chain reaction.
p53 gene
Bax gene
Caspase-9 gene
Cytochrome c gene
b-Actin gene
Primer sequences
Annealing Tm
5-TATGAGCATCGAGCTC CCTCT-3 5-CACAACTGCACAGGGC ATGT-3 5-AGACAGGGGCCTTTTT GTTAC-3 5-GAGGACTCCAGCCACA AAGAT-3 5-GTAAACTTTGGCGGAC TG-3 5-AGCCTCTGAAATAGCA CC-3 5-TTTGGATCCAATGGGT GATGTTGAG-3 5-TTTGAATTCCTCATTA GTAGCTTTTTTGAG-3 5-CCAGGCTGGATTGCA GTT-3 5-GATCACGAGGTCAGG AGATG-3
58 C
58 C
56 C
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Table II. Effect of cisplatin and spermatogonial stem cell transplantation on biochemical parameters in experimental and control groups of male rats.
Control MDA (nmol/g of 29.7 wet tissue) Catalase (U/g of 23.8 wet tissue) GSH-Px (U/g of 7.3 wet tissue) Testosterone (ng/dL) 330.5
Cisplatin
Cisplatin þ stem cell
2.7
45.7 4.2a 30.3 2.4b
3.7
15.6 4.8a 21.4 1.9a,b
1.7
4.8 0.3a
6.7 1.1b
39
161.9 25a
212 32a,b
P < 0.01, control versus other groups. P < 0.001, cisplatin versus cisplatin þ stem cell.
a
b
56 C
55 C
for 15 minutes. The supernatant was used for the assay. The test is based on the determination of the H2O2 decomposition rate at 240 nm. Results are expressed in units per gram of tissue. Determination of glutathione peroxidase activity GSH-Px activity (GSH-Px) was measured according to Wendel [15]. The testis was homogenized at 4 C in the ratio of 1:10 in phosphate buffer (pH 7.0) and centrifuged at 3500 rpm for 15 min. The enzyme reaction in the tube, which contains sodium azide, NADPH, reduced glutathione, and glutathione reductase, was initiated by addition of hydrogen peroxide and the change in absorbance at 340 nm. Activity was given as units per gram of tissue.
deoxyribonucleoside triphosphate (dNTP) mixtures and 0.5 mL of AMV reverse transcriptase. The mixture was incubated at 37 C for 10 min, 52 C for 45 min, 95 C for 5 min and ice path for 5 min. The relative abundance of messenger RNA (mRNA) species was assessed with the use of the SYBR Green method on an ABI prism 7500 sequence detector system (Applied Biosystems). The sequences of the oligonucleotide primers used in these assays are shown in Table I. Quantitative real-time polymerase chain reaction (PCR) was performed in duplicate in a 25-mL reaction volume consisting of 2X SYBR Green PCR Master Mix (Applied Biosystems), 900 nmol/L of each primer and 2 to 3 mL of cDNA. Amplification conditions were 2 min at 50 C, 10 min at 95 C and 40 cycles of denaturation for 15 s and annealing/ extension at 60 C for 10 min. Data from real-time assays were calculated with the use of version 1.7 Sequence Detection Software from PE Biosystems. Relative expression of p53, Bax, caspase and cytochrome c mRNA was calculated by use of the comparative Ct method as previously described [16]. All values were normalized to the b-actin mRNA.
Determination of plasma testosterone levels The plasma testosterone level was measured with the use of an enzyme-linked immunosorbent assay testosterone kit according to the kit manufacturer’s instructions and expressed as ng/mL. Real-time quantitative analyses for gene expression of P53, Bax, caspase 9 and cytochrome c RNA was isolated with the use of Trizol reagent, by use of the protocol provided by the manufacturer. The RNA was reverse-transcribed in a 10-mL mixture containing 6 mL of total RNA, 0.5 mL random primers (Promega), 2 mL of 5 reversetranscriptase buffer (Life Technology), 1 mL of
Figure 1. Effect of CP and spermatogonial stem cell on gene expression in experimental and control groups of male rats.
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Figure 2. Effect of CP and spermatogonial stem cells on sperm characteristics in experimental and control groups of male rats. (A) Testicular weight (g), (B) area% of collagen fibers, (C) sperm count (106/mL), (D) sperm motility (%) and sperm abnormality (%), (E) diameter of seminiferous tubule (mm) and germinal epithelial height (mm). *P <0.001 Control vs Cisplatin; #P <0.001 Cisplatin vs Cisplatin þ Stem cell.
Sperm motility, sperm count and sperm morphological abnormalities Briefly, right epididymal sperms were collected in 5 mL of Ham’s F10 and incubated for 5 min at 37 C in the atmosphere of 5% CO2. The sperm count was determined with the use of a hemocytometer. One
drop of sperm suspension was placed into a counting chamber. The total sperm count in eight squares of 1 mm2 each was determined and multiplied by 5 104 to get the total count. Sperm motility was counted in the same eight squares and percentage of motility was recorded [17]. For the analysis of
Spermatogonial stem cells
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Figure 2. (continued).
morphological abnormalities, sperm smears were drawn on slides and the slides were stained with 1% eosineY/5% nigrosin and examined at magnification 400 for morphological abnormalities such as amorphous, hook less, bicephalic and coiled or abnormal tails [17]. Histopathologic examinations At the time of euthanasia, the rats were anesthetized with ether inhalation. After the testes were weighed, they were fixed in Bouin’s solution for 24 h and were processed to prepare 5-mm-thick paraffin sections for hematoxylin and eosin (H&E) and Masson’s trichrome stains. The data were obtained with the use of the image analyzer computer system (Leica Qwin 500). The mean of 50 different fields per slide used for the result. The diameter of the seminiferous tubules (STs) and the height of their lining epithelium were measured in 10 low-power fields (LPF) with the use of the interactive measurements menu. The area % of collagen fibers was measured in 10 LPF.
Statistical analysis The results for quantitative variables are expressed as mean standard deviation. The means of the three genotype groups were compared in a one-way analysis of variance and multiple comparison post hoc tests. Differences between groups were considered significant when P values were <0.05. All statistical analyses were performed with the use of SPSS, version 10 (SPSS Inc). Results Effects of CP and spermatogonial stem cells on biochemical parameters in studied groups In comparison with the control group, CP administration alone showed a significant increase in MDA levels (P < 0.001). Stem cell administration to CPtreated rats reduced these increased MDA levels (P < 0.001). A significant decrease in CAT and GSH-Px activities was demonstrated in CP-treated animals as compared with control, which was
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Figure 3. H&E-stained section of testes of experimental groups. (A,B) Control group, showing regular seminiferous tubules (St), narrow inter-tubular space (*). Tubules are lined by stratified germinal epithelium with spermatogonia (1), spermatocytes (2), round spermatid (3), elongated spermatid (4) and sperms in the lumina of tubules (Lu). Oval pale nuclei of Sertoli cells (s) appear. Clusters of Leydig cells (L) were observed around a blood capillary (c). (CeF) Cisplatin-treated rats, showing many distorted disorganized and shrunken seminiferous tubules (St) separated by a wide interstitium space (s) in between. Some tubules show marked reduction in the thickness of the germinal epithelium (arrows) and decreased/or no sperms, whereas others have exfoliated germ cells (double arrows) in their lumina and vacuoles (v). Some seminiferous tubules show irregular basement membranes (arrowheads) (C, D). Darkly stained nuclei (arrows) of the basal spermatogonia (arrowheads) are also seen in most of tubules (E). Multi-nuclear giant cells (curved arrow) are shown in the lumen of the tubules (F). (G, H) Seminiferous tubules of the stem cell recipient group. The adjacent tubules (St) are lined with normal stratification of germinal epithelial (double-head arrow) with normal appearance of the interstitial cells in the interstitial tissue (*). Few spermatogenic cells showed vacuolated cytoplasm (arrowheads). The tubular lumen (Lu) is filled with sperms (A, C, D and G, magnification 100) (B, E, F and H, magnification 400) (scale bar ¼ 50 mm).
significantly restored by stem cell treatment. CP administration decreased the plasma testosterone levels significantly (P < 0.01) when compared with
the control group. However, plasma testosterone levels were elevated significantly (P < 0.01) by stem cell administration (Table II).
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Figure 4. Masson’s trichromeestained section of testes of experimental groups. (A) Control group, showing fine collagen fibers at the boundary tissue of seminiferous tubules (arrow) and in between the tubules (*). (B, C) CP-treated rats, showing dense collagen fibers at the boundary tissue of seminiferous tubules (arrow), around blood vessels (arrowhead) and in between the tubules (*). (D) Seminiferous tubules of stem cell co-treatment group showing fine collagen fibers at the boundary tissue of seminiferous tubules (arrow) and in between the tubules (*) (AeD, magnification 400) (scale bar ¼ 50 mm).
Effects of cisplatin and spermatogonial stem cells on gene expression in studied groups A significant decrease in p53 gene expression levels was demonstrated in CP-treated animals as compared with the control, which was significantly restored by stem cell treatment. In comparison
with the control group, CP administration alone caused a significant increase in Bax, cytochrome c and caspase 9 gene expression levels (P < 0.001). Stem cell administration to CP-treated rats reduced these elevated levels (P < 0.001) (Figure 1).
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Effects of CP and spermatogonial stem cells on sperm characteristics in studied groups Treatment of male rats with CP caused a significant decrease in the testicular weight, diameter of seminiferous tubules, height of the germinal epithelial lining of the seminiferous tubules, sperm count and motility (P < 0.001) and a significant increase in collagen fibers and sperm abnormality as compared with control. Concomitant administration of stem cell with CP caused a significant improvement in all parameters and minimized the toxic effects of CP (P < 0.001) (Figure 2). Histopathological examination of the testes H&E-stained sections of specimens removed from the control (Figure 3A,B) were normal, with no pathological changes. Testicular sections of the CPtreated rats showed severe degenerative changes characterized by disorganized seminiferous tubules confirming impaired spermatogenesis. Wide separation of inter-tubular space was observed. Most of tubules lined by atrophied germinal epithelium with marked reduction in height. Some tubules showed multiple small dark nuclei and/or vacuolated cytoplasm of spermatogenic cells and decreased sperms (Figure 3C-F). Stem cell co-treatment showed preserved testicular morphology, in which the STs appeared normal. Few spermatogenic cells showed a vacuolated cytoplasm (Figure 3G,H; also see Figure 3). Examination of Masson’s trichromeestained sections of specimens removed from the control group showed fine collagen fibers at the boundary tissue of seminiferous tubules, around blood vessels and in between the tubules (Figure 4A). Testicular sections of the CP-treated rats showed dense collagen fibers at the boundary tissue of seminiferous tubules, around blood vessels and in between the tubules (Figure 4B,C). Stem cell co-treatment showed fine collagen fibers at the boundary tissue of seminiferous tubules, around blood vessels and in between the tubules (Figure 4D; also see Figure 4). Discussion Treatment with cancer chemotherapy is associated with significant gonadal damage in the male reproductive organs [18]. The cisplatin-induced testicular damage in animals is commonly associated with spermatogenic damage, germ cell apoptosis, Leydig cell dysfunction and testicular steroidogenic disorder [19]. In the current study, after CP administration; we found an increase of testicular MDA levels and a decrease in CAT and GSH-Px activities. Our results
are in agreement with others [20,21]. Pathogenesis of testicular damage and gonadotoxicity after CP exposure is ascribed to direct interaction with DNA. CP was suggested to generate hydroxyl radicals and superoxide anion and subsequently increase lipid peroxidation and decrease the activity of anti-oxidant enzymes [2,22,23]. A decrease in the activities of anti-oxidants enzymes probably was due to their overuse in chemical reactions that were meant to neutralize the MDA or due to suppression of their synthesis by oxidative stress [5,6,23]. The results of the current study provide that administration of SCC after CP therapy protects the testes against CP testicular toxicity through oxidative stress suppression and significant increase in the activities of anti-oxidant enzymes. The decline of lipid peroxidation indicates that spermatogonial stem cells potently scavenged the free radicals and suppressed oxidative DNA damage; this supports the hypothesis that the mechanism of testicular damage could be attributed, at least in part, to the overproduction of free radicals. In our study, CP injection resulted in a decreased expression of p53 and an increase the expression of Bax, cytochrome c and caspase 9 (the intrinsic pathway of the cell death). Also, we showed that administration of SSC led to a significant increase in gene expression of P53 and a decrease in expression of Bax, cytochrome c and caspase 9 genes. It has been reported that acute and chronic exposure to CP results in elevated apoptotic germ cell rates [5,24,25]. A caspase-dependent pathway could be triggered by chemotherapeutics [26]. In addition, fatty acid oxidation products do affect signal transduction pathways, particularly those involved in apoptosis [27]. Our study showed that the spermatotoxic effects of the CP led to diminished sperm motility, possibly as the result of impaired tail function, decrease in sperm count caused by cytotoxicity and a significant decrease in plasma testosterone caused by Leydig cell dysfunction [28]. Our results are in agreement with others [2,6,29]. Administration of SSC in CPtreated rats significantly increased the testosterone levels. We suggest that the increase of serum testosterone levels is attributed to the anti-oxidant activity of SCC, which protects Leydig cells from oxidative damage. The negative changes observed in sperm quality after CP exposure in the present study may be attributed to the high concentration of MDA and low anti-oxidant capacity and peroxidation of polyunsaturated fatty acids in membranes of spermatozoa, a process resulting in reduced sperm viability and motility and thus infertility [30]. The results of the current study showed that administration of SCC could significantly reduce the
Spermatogonial stem cells increased MDA levels in comparison to the only CP group in this work. The decline of lipid peroxidation in testicular tissue apparently indicates that SCC scavenged the free radicals and suppressed oxidative DNA damage. This status indicates that SCC has potent anti-oxidant activity. Also, we showed that administration of SSC led to a significant increase in gene expression of P53 and a decrease in expression of Bax, cytochrome c and caspase 9 genes through potent anti-apoptotic properties. Conclusions The current study highlights evidence that the SCC has anti-oxidative and anti-apoptotic properties that could reverse CP-induced testicular toxicity, in addition to their role in spermatogenesis. Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. References [1] Amin A, Buratovich M. New platinum and ruthenium complexes—the latest class of potential chemotherapeutic drugs—a review of recent developments in the field. Mini Rev Med Chem 2009;9:1489e503. [2] Atessahin A, Sahna E, Türk G, Ceribasi AO, Yilmaz S, Yüce A, et al. Chemoprotective effect of melatonin against Cisplatininduced testicular toxicity in rats. J Pineal Res 2006;41:21e7. [3] Fung C, Vaughn DJ. Complications associated with chemotherapy in testicular cancer management. Nat Rev Urol 2011;8:213e22. [4] Hooser SB, van Dijk-Knijnenburg WC, WaalkensBerendsen ID, Smits-van Prooije AE, Snoeij NJ, Baan RA, et al. Cisplatin-DNA adducts formation in rat spermatozoa and its effects on fetal development. Cancer Lett 2000;151: 71e80. [5] Amin A, Hamza AA, Kambal A, Daoud S. Herbal extracts counteract Cisplatin-mediated cell death in rat testis. Asian J Androl 2008;10:291e7. [6] Türk G, Atessahin A, Sönmez M, Ceribasi AO, Yüce A. Improvement of Cisplatin-induced injuries to sperm quality, the oxidanteantioxidant system, and the histologic structure of the rat testis by ellagic acid. Fertil Steril 2008;89:1474e81. [7] Agarwal A, Cocuzza M, Abdelrazik H, Sharma RK. Oxidative stress measurement in patients with male or female factor infertility. In: Popov I, Lewin G. Handbook of Chemiluminescent Methods in Oxidative Stress Assessment. Kerala, India: Transworld Research Network; 2008. p. 195e218. [8] Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci USA 1994;91:11298e302. [9] Nagano M, Avarbock MR, Brinster RL. Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient tests. Biol Reprod 1999;60:1429e36. [10] Aslam I, Fishel S, Moore H, Dowell K, Thornton S. Fertility preservation of boys undergoing anti-cancer therapy: a review of the existing situation and prospects for the future. Hum Reprod 2000;15:2154e9.
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[11] Nayernia K, Li M, Engel W. Spermatogonial Stem Cells. Methods Mol Biol 2004;253:105e20. [12] Pogach LM, Lee Y, Gould S, Giglio W, Meyenhofer M, Huang HF. Characterization of cis-platinum-induced Sertoli cell dysfunction in rodents. Toxicol Appl Pharmacol 1989; 98:350e61. [13] Ohkawa H, Ohishi N, Yagi K. Assay for Lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351e8. [14] Aebi H. Catalase. In: Bergmeyer HU. Methods of enzymatic analysis. Chemic Academic Press Inc Verlag; 1974. p. 673e85. [15] Wendel A. Glutathione peroxidase. Methods Enzymol 1981; 77:325e33. [16] Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nat Protoc 2008;3:1101e8. [17] World Health Organization. WHO Laboratory Manual for the Examination 62 and Processing of Human Semen. 5th ed. Geneva, CH: World Health Organization; 2010. [18] Çeribas¸ı AO, Sakin F, Türk G, Sönmez M, Ates¸s¸ahin A. Impact of ellagic acid on adriamycin-induced testicular histopathological lesions, apoptosis, lipid peroxidation and sperm damages. Exp Toxicol Pathol 2012;64:717e24. [19] Cherry SM, Hunt PA, Hassold TJ. Cisplatin disrupts mammalian spermatogenesis, but does not affect recombination or chromosome segregation. Mutat Res 2004;564: 115e28. [20] Silici S, Ekmekcioglu O, Eraslan G, Demirtas A. Antioxidative effect of royal jelly in Cisplatin-induced testes damage. Urology 2009;74:545e51. [21] Amin A, Hamza AA. Effects of Roselle and Ginger on cisplatin-induced reproductive toxicity in rats. Asian J Androl 2006;8:607e12. [22] Ahmed EA, Omar HM, elghaffar SKh, Ragb SMM, Nasser AY. The antioxidant activity of Vitamin C, DPPD and L-cysteine against Cisplatin-induced testicular oxidative damage in rats. Food Chem Toxicol 2011;49:1115e21. [23] Masuda H, Tanaka T, Takahama U. Cisplatin generates superoxide anion by interaction with DNA in a cell-free system. Biochem Biophys Res Commun 1994;203:1175e80. [24] Ilbey YO, Ozbek E, Simsek A, Otunctemur A, Cekmen M, Somay A. Potential chemoprotective effect of melatonin in cyclophosphamide- and Cisplatin induced testicular damage in rats. Fertil Steril 2009;92:1124e32. [25] Mansilla S, Llovera L, Portugal J. Chemotherapeutic targeting of cell death pathways. Anticancer Agents Med Chem 2012;12:226e38. [26] Tang DG, La E, Kern J, Kehrer JP. Fatty acid oxidation and signaling in apoptosis. Biol Chem 2002;383:425e42. [27] Lirdi LC, Stumpp T, Sasso-Cerri E, Miraglia SM. Amifostine protective effect on cisplatin-treated rat testis. Anat Rec 2008;291:797e808. [28] Pectasides D, Pectasides M, Farmakis D, Nikolaou M, Koumpou M, Kostopoulou V, et al. Testicular function in patients with testicular cancer treated with Bleomycine EtoposideeCarboplatin (BEC90) combination chemotherapy. Eur Urol 2004;45:187e93. [29] Ilbey YO, Ozbek E, Simsek A, Cekmen M, Otunctemur A, Somay A. Chemoprotective Effect of a Nuclear Factor-kB Inhibitor, Pyrrolidine Dithiocarbamate, Against CisplatinInduced Testicular Damage in Rats. J Androl 2009;30: 505e14. [30] Aitken RJ, Curry BJ. Redox regulation of human sperm function: from the physiological control of sperm capacitation to the etiology of infertility and DNA damage in the germ line. Antioxid Redox Signal 2011;14:367e81.
Anti-oxidative and anti-apoptotic roles of spermatogonial stem cells in reversing cisplatin-induced testicular toxicity
YOUSRI M. HUSSEIN1, RANDA H. MOHAMED2, SALLY M. SHALABY2, MANAL R. ABD EL-HALEEM3 & DALIA M. ABD EL MOTTELEB4 1
Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt, Medical Laboratory Department, Faculty of Applied Medical Science, Taif University, Taif, KSA, 2Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt, 3Histology and Cell Biology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt, Histology Department, Faculty of Oral Medicine and Dentistry, MTI University, Cairo, Egypt and 4Clinical Pharmacology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt. Abstract Background aims. Because of reproductive toxic effects of chemotherapy, researchers have taken some techniques to preserve fertility potential. The present study was designed to point out the potential role of spermatogonial stem cell (SSC) therapy in reversing cisplatin (CP)-induced testicular toxicity and restore the spermatogenesis. Methods. Sixty rats were randomly divided into three groups: group 1, control group; group 2, rats received CP in a dose of 7 mg/kg/day for 5 consecutive days; group 3, CP was injected at 7 mg/kg per day for 5 consecutive days, and, on the 6th day of the experiment, rats were treated with SSC. Forty days after receiving the last dose of CP, rats were euthanized under anesthesia; testes were collected, and gene expression using real-time polymerase chain reaction for P53, Bax, caspase 9 and cytochrome c, testicular histological findings and oxidative status were determined. Results. Administration of cisplatin caused significant increases in malondialdehyde levels, Bax and caspase 9 genes expression levels concomitant with significant decreases in anti-oxidant enzyme activities, p53 and cytochrome c gene expression levels, along with some histopathological lesions in testicular tissue. SCC attenuated the disturbance in oxidant/anti-oxidant status and testicular apoptosis; this is associated with improvements in the histopathological view of the testicular tissue. Conclusions. The current study highlights evidence that the SCC has antioxidative and anti-apoptotic properties that could reverse CP-induced testicular toxicity, in addition to their role in spermatogenesis. Key Words: anti-apoptotic, anti-oxidant, cisplatin, infertility, spermatogonial stem cell
Introduction Cisplatin (CP-diamminedichloroplatinum-II, CP) is a highly effective chemotherapeutic agent used for the treatment of many types of cancers including sarcomas, lymphomas, testicular, ovarian, breast, lung and bladder cancer [1,2]. Despite the broadspectrum clinical applications of CP, severe toxic side effects such as reproductive toxicity and increased the frequency of germ cell apoptosis, constitute a major consequence to its use [3,4]. The precise mechanism by which CP causes testicular toxicity and germ cell apoptosis is not fully known; however, numerous studies have shown that CP exposure can disrupt the redox balance of tissues, suggesting that biochemical and physiological disturbances may result from oxidative stress [5,6].
Reactive oxygen species are normally generated in mitochondria of testes, which are subsequently scavenged by anti-oxidant defense systems of the corresponding cellular compartments [7]. However, this balance can easily be broken by chemicals such as cisplatin, which disrupt the pro-oxidanteantioxidant balance, leading to cellular dysfunction [6]. Researchers have used some techniques such as the development of a spermatogonial transplantation, which provided a new treatment strategy for male infertility after the use of chemotherapy and irradiation [8]. The spermatogonial stem cells (SSC) colonize and initiate spermatogenesis, after the transplantation of dissociated testis cells into a seminiferous tubule microenvironment [9]. Because SSC self-renew and differentiate into proliferating spermatogonia, they
Correspondence: Randa H. Mohamed, MD, Faculty of Medicine, Zagazig University, 44519 Zagazig, Egypt. E-mail: [email protected] (Received 29 January 2015; accepted 5 July 2015) ISSN 1465-3249 Copyright Ó 2015, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2015.07.001
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provide a limitless supply of mature spermatozoa. Thus, SSC transplantation may be useful for the treatment of male infertility. It may also offer a great help in cancer patients who are undergoing stem celldestroying irradiation or chemotherapy, by prior isolation of stem cells and autotransplantation [10]. The purpose of the present study was to assess the potential role of stem cell therapy in reversing cisplatin-induced testicular toxicity and on antioxidant and anti-apoptotic capacity. Methods Chemicals Cisplatin (10 mg/10 mL) was from Sigma-Aldrich Inc. The other chemicals were obtained from Sigma. Preparation of donor cells and transplantation Donor testes were prepared according to Nayernia et al. [11]. Testes were isolated from four or five white albino rats (age between 5e10 days), washed in phosphate-buffered saline, and put in 5 mL of collagenase/Hanks balanced salt solution (pH 7.4) in a 60-mm sterile Petri dish. It was then incubated for 10 to 15 min at 37 C with gentle agitation on a rocker platform until the tubules separated. The tubules were poured off and transferred to a 10-mL cell culture centrifugation tube and centrifuged at 4 C at 650g for 5 min. The supernatant was added to 5 mL of Hanks’ balanced salt solution and centrifuged again at 4 C at 650g for 5 min. Dispase solution (2 mL) was added and stirred slowly at 37 C until the tissue was sufficiently dissolved (30 min). Dispersion of the tubule cells can be hastened by pipetting and gentle agitation. Fresh dispase solution was added to the remaining tubule fragment if further disaggregation is required. Cells were pelleted by centrifugation at 16 C at 650g for 5 min. The pellet was resuspended in 400 mL of Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). The cells can be maintained for 1 to 5 h at 4 C until transplantation. Trypan blue (0.3%) was added, and the cells were transplanted into the seminiferous tubules through the efferent ductules of recipient rat.
ad libitum access to standard rodent chow diet and filtered water. The rats were allowed to acclimate for 1 week before any use in experiments. All experimental procedures were approved by the local authorities, that is, the Ethical Committee for Animal Handling at Zagazig University, and were conducted according to the guidelines set forth by the National Institutes of Health (USA). For the experiment, rats were divided into three groups containing 20 rats per group and received the following: the control group received 10 mL of DMEM containing 10% FBS and trypan blue (0.3%) (vehicle of SSC) through injection into the testes for 6 consecutive days of the experiment. Group 2 rats were treated with CP alone: CP was injected (intra-peritoneally) in a dose of 7 mg/kg per day for 5 consecutive days. On the 6th day of the experiment, injections of 10 mL of DMEM containing 10% FBS and trypan blue (0.3%) (vehicle of SSC) to the testes via inserting a needle through the efferent ductules outside of the testis and passing the needle into the rete testis. In group 3, CP was injected intra-peritoneally at 7 mg/kg per day for 5 consecutive days. On the 6th day of experiment, rats received 1 106 cells SSC dissolved in 10 mL of DMEM containing 10% FBS and trypan blue (0.3%) in a single dose via inserting a needle through the efferent ductules outside of the testis and passing the needle into the rete testis. The testicular toxicity of CP can be detected within 7 weeks after treatment with a sufficient dose in rats [12]. The rats were killed under anesthesia (ketamine, 200 mg/kg intraperitoneally) at the end of the treatment period. Biochemical analysis For determination of lipid peroxidation levels, testicular tissue lipid peroxide levels (as malondialdehyde, MDA) were measured with the use of the thiobarbituric acid (TBA) reaction method of Ohkawa et al. [13]. The testis was decapsulated and homogenized in 10% trichloroacetic acid. MDA reacted with freshly prepared TBA to form a colored complex, and the absorption of the supernatant was recorded at 532 nm by comparing the absorption with the standard curve of MDA equivalents generated by acid-catalyzed hydrolysis of 1,1,3,3tetramethoxypropane.
Animals and experimental design Male Wistar rats (n ¼ 60; 8e10 weeks old; 200e220 g) were obtained from the Faculty of Veterinary Medicine at Zagazig University. On arrival, rats were housed under constant environmental conditions (room temperature 25 2 C with a 12-h light/dark cycle and 50% humidity). Rats were provided
Anti-oxidant enzyme assay Catalase activity (CAT) was determined according to the method of Aebi [14]. The testis was homogenized at 4 C in potassium phosphate buffer (50 mmol/L potassium phosphate and 1 mmol/L ethylenediamine tetra-acetic acid, pH 7) and centrifuged at 3500 rpm
Spermatogonial stem cells Table I. Sequence of the primers used for real-time polymerase chain reaction.
p53 gene
Bax gene
Caspase-9 gene
Cytochrome c gene
b-Actin gene
Primer sequences
Annealing Tm
5-TATGAGCATCGAGCTC CCTCT-3 5-CACAACTGCACAGGGC ATGT-3 5-AGACAGGGGCCTTTTT GTTAC-3 5-GAGGACTCCAGCCACA AAGAT-3 5-GTAAACTTTGGCGGAC TG-3 5-AGCCTCTGAAATAGCA CC-3 5-TTTGGATCCAATGGGT GATGTTGAG-3 5-TTTGAATTCCTCATTA GTAGCTTTTTTGAG-3 5-CCAGGCTGGATTGCA GTT-3 5-GATCACGAGGTCAGG AGATG-3
58 C
58 C
56 C
3
Table II. Effect of cisplatin and spermatogonial stem cell transplantation on biochemical parameters in experimental and control groups of male rats.
Control MDA (nmol/g of 29.7 wet tissue) Catalase (U/g of 23.8 wet tissue) GSH-Px (U/g of 7.3 wet tissue) Testosterone (ng/dL) 330.5
Cisplatin
Cisplatin þ stem cell
2.7
45.7 4.2a 30.3 2.4b
3.7
15.6 4.8a 21.4 1.9a,b
1.7
4.8 0.3a
6.7 1.1b
39
161.9 25a
212 32a,b
P < 0.01, control versus other groups. P < 0.001, cisplatin versus cisplatin þ stem cell.
a
b
56 C
55 C
for 15 minutes. The supernatant was used for the assay. The test is based on the determination of the H2O2 decomposition rate at 240 nm. Results are expressed in units per gram of tissue. Determination of glutathione peroxidase activity GSH-Px activity (GSH-Px) was measured according to Wendel [15]. The testis was homogenized at 4 C in the ratio of 1:10 in phosphate buffer (pH 7.0) and centrifuged at 3500 rpm for 15 min. The enzyme reaction in the tube, which contains sodium azide, NADPH, reduced glutathione, and glutathione reductase, was initiated by addition of hydrogen peroxide and the change in absorbance at 340 nm. Activity was given as units per gram of tissue.
deoxyribonucleoside triphosphate (dNTP) mixtures and 0.5 mL of AMV reverse transcriptase. The mixture was incubated at 37 C for 10 min, 52 C for 45 min, 95 C for 5 min and ice path for 5 min. The relative abundance of messenger RNA (mRNA) species was assessed with the use of the SYBR Green method on an ABI prism 7500 sequence detector system (Applied Biosystems). The sequences of the oligonucleotide primers used in these assays are shown in Table I. Quantitative real-time polymerase chain reaction (PCR) was performed in duplicate in a 25-mL reaction volume consisting of 2X SYBR Green PCR Master Mix (Applied Biosystems), 900 nmol/L of each primer and 2 to 3 mL of cDNA. Amplification conditions were 2 min at 50 C, 10 min at 95 C and 40 cycles of denaturation for 15 s and annealing/ extension at 60 C for 10 min. Data from real-time assays were calculated with the use of version 1.7 Sequence Detection Software from PE Biosystems. Relative expression of p53, Bax, caspase and cytochrome c mRNA was calculated by use of the comparative Ct method as previously described [16]. All values were normalized to the b-actin mRNA.
Determination of plasma testosterone levels The plasma testosterone level was measured with the use of an enzyme-linked immunosorbent assay testosterone kit according to the kit manufacturer’s instructions and expressed as ng/mL. Real-time quantitative analyses for gene expression of P53, Bax, caspase 9 and cytochrome c RNA was isolated with the use of Trizol reagent, by use of the protocol provided by the manufacturer. The RNA was reverse-transcribed in a 10-mL mixture containing 6 mL of total RNA, 0.5 mL random primers (Promega), 2 mL of 5 reversetranscriptase buffer (Life Technology), 1 mL of
Figure 1. Effect of CP and spermatogonial stem cell on gene expression in experimental and control groups of male rats.
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Figure 2. Effect of CP and spermatogonial stem cells on sperm characteristics in experimental and control groups of male rats. (A) Testicular weight (g), (B) area% of collagen fibers, (C) sperm count (106/mL), (D) sperm motility (%) and sperm abnormality (%), (E) diameter of seminiferous tubule (mm) and germinal epithelial height (mm). *P <0.001 Control vs Cisplatin; #P <0.001 Cisplatin vs Cisplatin þ Stem cell.
Sperm motility, sperm count and sperm morphological abnormalities Briefly, right epididymal sperms were collected in 5 mL of Ham’s F10 and incubated for 5 min at 37 C in the atmosphere of 5% CO2. The sperm count was determined with the use of a hemocytometer. One
drop of sperm suspension was placed into a counting chamber. The total sperm count in eight squares of 1 mm2 each was determined and multiplied by 5 104 to get the total count. Sperm motility was counted in the same eight squares and percentage of motility was recorded [17]. For the analysis of
Spermatogonial stem cells
5
Figure 2. (continued).
morphological abnormalities, sperm smears were drawn on slides and the slides were stained with 1% eosineY/5% nigrosin and examined at magnification 400 for morphological abnormalities such as amorphous, hook less, bicephalic and coiled or abnormal tails [17]. Histopathologic examinations At the time of euthanasia, the rats were anesthetized with ether inhalation. After the testes were weighed, they were fixed in Bouin’s solution for 24 h and were processed to prepare 5-mm-thick paraffin sections for hematoxylin and eosin (H&E) and Masson’s trichrome stains. The data were obtained with the use of the image analyzer computer system (Leica Qwin 500). The mean of 50 different fields per slide used for the result. The diameter of the seminiferous tubules (STs) and the height of their lining epithelium were measured in 10 low-power fields (LPF) with the use of the interactive measurements menu. The area % of collagen fibers was measured in 10 LPF.
Statistical analysis The results for quantitative variables are expressed as mean standard deviation. The means of the three genotype groups were compared in a one-way analysis of variance and multiple comparison post hoc tests. Differences between groups were considered significant when P values were <0.05. All statistical analyses were performed with the use of SPSS, version 10 (SPSS Inc). Results Effects of CP and spermatogonial stem cells on biochemical parameters in studied groups In comparison with the control group, CP administration alone showed a significant increase in MDA levels (P < 0.001). Stem cell administration to CPtreated rats reduced these increased MDA levels (P < 0.001). A significant decrease in CAT and GSH-Px activities was demonstrated in CP-treated animals as compared with control, which was
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Figure 3. H&E-stained section of testes of experimental groups. (A,B) Control group, showing regular seminiferous tubules (St), narrow inter-tubular space (*). Tubules are lined by stratified germinal epithelium with spermatogonia (1), spermatocytes (2), round spermatid (3), elongated spermatid (4) and sperms in the lumina of tubules (Lu). Oval pale nuclei of Sertoli cells (s) appear. Clusters of Leydig cells (L) were observed around a blood capillary (c). (CeF) Cisplatin-treated rats, showing many distorted disorganized and shrunken seminiferous tubules (St) separated by a wide interstitium space (s) in between. Some tubules show marked reduction in the thickness of the germinal epithelium (arrows) and decreased/or no sperms, whereas others have exfoliated germ cells (double arrows) in their lumina and vacuoles (v). Some seminiferous tubules show irregular basement membranes (arrowheads) (C, D). Darkly stained nuclei (arrows) of the basal spermatogonia (arrowheads) are also seen in most of tubules (E). Multi-nuclear giant cells (curved arrow) are shown in the lumen of the tubules (F). (G, H) Seminiferous tubules of the stem cell recipient group. The adjacent tubules (St) are lined with normal stratification of germinal epithelial (double-head arrow) with normal appearance of the interstitial cells in the interstitial tissue (*). Few spermatogenic cells showed vacuolated cytoplasm (arrowheads). The tubular lumen (Lu) is filled with sperms (A, C, D and G, magnification 100) (B, E, F and H, magnification 400) (scale bar ¼ 50 mm).
significantly restored by stem cell treatment. CP administration decreased the plasma testosterone levels significantly (P < 0.01) when compared with
the control group. However, plasma testosterone levels were elevated significantly (P < 0.01) by stem cell administration (Table II).
Spermatogonial stem cells
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Figure 4. Masson’s trichromeestained section of testes of experimental groups. (A) Control group, showing fine collagen fibers at the boundary tissue of seminiferous tubules (arrow) and in between the tubules (*). (B, C) CP-treated rats, showing dense collagen fibers at the boundary tissue of seminiferous tubules (arrow), around blood vessels (arrowhead) and in between the tubules (*). (D) Seminiferous tubules of stem cell co-treatment group showing fine collagen fibers at the boundary tissue of seminiferous tubules (arrow) and in between the tubules (*) (AeD, magnification 400) (scale bar ¼ 50 mm).
Effects of cisplatin and spermatogonial stem cells on gene expression in studied groups A significant decrease in p53 gene expression levels was demonstrated in CP-treated animals as compared with the control, which was significantly restored by stem cell treatment. In comparison
with the control group, CP administration alone caused a significant increase in Bax, cytochrome c and caspase 9 gene expression levels (P < 0.001). Stem cell administration to CP-treated rats reduced these elevated levels (P < 0.001) (Figure 1).
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Effects of CP and spermatogonial stem cells on sperm characteristics in studied groups Treatment of male rats with CP caused a significant decrease in the testicular weight, diameter of seminiferous tubules, height of the germinal epithelial lining of the seminiferous tubules, sperm count and motility (P < 0.001) and a significant increase in collagen fibers and sperm abnormality as compared with control. Concomitant administration of stem cell with CP caused a significant improvement in all parameters and minimized the toxic effects of CP (P < 0.001) (Figure 2). Histopathological examination of the testes H&E-stained sections of specimens removed from the control (Figure 3A,B) were normal, with no pathological changes. Testicular sections of the CPtreated rats showed severe degenerative changes characterized by disorganized seminiferous tubules confirming impaired spermatogenesis. Wide separation of inter-tubular space was observed. Most of tubules lined by atrophied germinal epithelium with marked reduction in height. Some tubules showed multiple small dark nuclei and/or vacuolated cytoplasm of spermatogenic cells and decreased sperms (Figure 3C-F). Stem cell co-treatment showed preserved testicular morphology, in which the STs appeared normal. Few spermatogenic cells showed a vacuolated cytoplasm (Figure 3G,H; also see Figure 3). Examination of Masson’s trichromeestained sections of specimens removed from the control group showed fine collagen fibers at the boundary tissue of seminiferous tubules, around blood vessels and in between the tubules (Figure 4A). Testicular sections of the CP-treated rats showed dense collagen fibers at the boundary tissue of seminiferous tubules, around blood vessels and in between the tubules (Figure 4B,C). Stem cell co-treatment showed fine collagen fibers at the boundary tissue of seminiferous tubules, around blood vessels and in between the tubules (Figure 4D; also see Figure 4). Discussion Treatment with cancer chemotherapy is associated with significant gonadal damage in the male reproductive organs [18]. The cisplatin-induced testicular damage in animals is commonly associated with spermatogenic damage, germ cell apoptosis, Leydig cell dysfunction and testicular steroidogenic disorder [19]. In the current study, after CP administration; we found an increase of testicular MDA levels and a decrease in CAT and GSH-Px activities. Our results
are in agreement with others [20,21]. Pathogenesis of testicular damage and gonadotoxicity after CP exposure is ascribed to direct interaction with DNA. CP was suggested to generate hydroxyl radicals and superoxide anion and subsequently increase lipid peroxidation and decrease the activity of anti-oxidant enzymes [2,22,23]. A decrease in the activities of anti-oxidants enzymes probably was due to their overuse in chemical reactions that were meant to neutralize the MDA or due to suppression of their synthesis by oxidative stress [5,6,23]. The results of the current study provide that administration of SCC after CP therapy protects the testes against CP testicular toxicity through oxidative stress suppression and significant increase in the activities of anti-oxidant enzymes. The decline of lipid peroxidation indicates that spermatogonial stem cells potently scavenged the free radicals and suppressed oxidative DNA damage; this supports the hypothesis that the mechanism of testicular damage could be attributed, at least in part, to the overproduction of free radicals. In our study, CP injection resulted in a decreased expression of p53 and an increase the expression of Bax, cytochrome c and caspase 9 (the intrinsic pathway of the cell death). Also, we showed that administration of SSC led to a significant increase in gene expression of P53 and a decrease in expression of Bax, cytochrome c and caspase 9 genes. It has been reported that acute and chronic exposure to CP results in elevated apoptotic germ cell rates [5,24,25]. A caspase-dependent pathway could be triggered by chemotherapeutics [26]. In addition, fatty acid oxidation products do affect signal transduction pathways, particularly those involved in apoptosis [27]. Our study showed that the spermatotoxic effects of the CP led to diminished sperm motility, possibly as the result of impaired tail function, decrease in sperm count caused by cytotoxicity and a significant decrease in plasma testosterone caused by Leydig cell dysfunction [28]. Our results are in agreement with others [2,6,29]. Administration of SSC in CPtreated rats significantly increased the testosterone levels. We suggest that the increase of serum testosterone levels is attributed to the anti-oxidant activity of SCC, which protects Leydig cells from oxidative damage. The negative changes observed in sperm quality after CP exposure in the present study may be attributed to the high concentration of MDA and low anti-oxidant capacity and peroxidation of polyunsaturated fatty acids in membranes of spermatozoa, a process resulting in reduced sperm viability and motility and thus infertility [30]. The results of the current study showed that administration of SCC could significantly reduce the
Spermatogonial stem cells increased MDA levels in comparison to the only CP group in this work. The decline of lipid peroxidation in testicular tissue apparently indicates that SCC scavenged the free radicals and suppressed oxidative DNA damage. This status indicates that SCC has potent anti-oxidant activity. Also, we showed that administration of SSC led to a significant increase in gene expression of P53 and a decrease in expression of Bax, cytochrome c and caspase 9 genes through potent anti-apoptotic properties. Conclusions The current study highlights evidence that the SCC has anti-oxidative and anti-apoptotic properties that could reverse CP-induced testicular toxicity, in addition to their role in spermatogenesis. Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. References [1] Amin A, Buratovich M. New platinum and ruthenium complexes—the latest class of potential chemotherapeutic drugs—a review of recent developments in the field. Mini Rev Med Chem 2009;9:1489e503. [2] Atessahin A, Sahna E, Türk G, Ceribasi AO, Yilmaz S, Yüce A, et al. Chemoprotective effect of melatonin against Cisplatininduced testicular toxicity in rats. J Pineal Res 2006;41:21e7. [3] Fung C, Vaughn DJ. Complications associated with chemotherapy in testicular cancer management. Nat Rev Urol 2011;8:213e22. [4] Hooser SB, van Dijk-Knijnenburg WC, WaalkensBerendsen ID, Smits-van Prooije AE, Snoeij NJ, Baan RA, et al. Cisplatin-DNA adducts formation in rat spermatozoa and its effects on fetal development. Cancer Lett 2000;151: 71e80. [5] Amin A, Hamza AA, Kambal A, Daoud S. Herbal extracts counteract Cisplatin-mediated cell death in rat testis. Asian J Androl 2008;10:291e7. [6] Türk G, Atessahin A, Sönmez M, Ceribasi AO, Yüce A. Improvement of Cisplatin-induced injuries to sperm quality, the oxidanteantioxidant system, and the histologic structure of the rat testis by ellagic acid. Fertil Steril 2008;89:1474e81. [7] Agarwal A, Cocuzza M, Abdelrazik H, Sharma RK. Oxidative stress measurement in patients with male or female factor infertility. In: Popov I, Lewin G. Handbook of Chemiluminescent Methods in Oxidative Stress Assessment. Kerala, India: Transworld Research Network; 2008. p. 195e218. [8] Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci USA 1994;91:11298e302. [9] Nagano M, Avarbock MR, Brinster RL. Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient tests. Biol Reprod 1999;60:1429e36. [10] Aslam I, Fishel S, Moore H, Dowell K, Thornton S. Fertility preservation of boys undergoing anti-cancer therapy: a review of the existing situation and prospects for the future. Hum Reprod 2000;15:2154e9.
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[11] Nayernia K, Li M, Engel W. Spermatogonial Stem Cells. Methods Mol Biol 2004;253:105e20. [12] Pogach LM, Lee Y, Gould S, Giglio W, Meyenhofer M, Huang HF. Characterization of cis-platinum-induced Sertoli cell dysfunction in rodents. Toxicol Appl Pharmacol 1989; 98:350e61. [13] Ohkawa H, Ohishi N, Yagi K. Assay for Lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351e8. [14] Aebi H. Catalase. In: Bergmeyer HU. Methods of enzymatic analysis. Chemic Academic Press Inc Verlag; 1974. p. 673e85. [15] Wendel A. Glutathione peroxidase. Methods Enzymol 1981; 77:325e33. [16] Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nat Protoc 2008;3:1101e8. [17] World Health Organization. WHO Laboratory Manual for the Examination 62 and Processing of Human Semen. 5th ed. Geneva, CH: World Health Organization; 2010. [18] Çeribas¸ı AO, Sakin F, Türk G, Sönmez M, Ates¸s¸ahin A. Impact of ellagic acid on adriamycin-induced testicular histopathological lesions, apoptosis, lipid peroxidation and sperm damages. Exp Toxicol Pathol 2012;64:717e24. [19] Cherry SM, Hunt PA, Hassold TJ. Cisplatin disrupts mammalian spermatogenesis, but does not affect recombination or chromosome segregation. Mutat Res 2004;564: 115e28. [20] Silici S, Ekmekcioglu O, Eraslan G, Demirtas A. Antioxidative effect of royal jelly in Cisplatin-induced testes damage. Urology 2009;74:545e51. [21] Amin A, Hamza AA. Effects of Roselle and Ginger on cisplatin-induced reproductive toxicity in rats. Asian J Androl 2006;8:607e12. [22] Ahmed EA, Omar HM, elghaffar SKh, Ragb SMM, Nasser AY. The antioxidant activity of Vitamin C, DPPD and L-cysteine against Cisplatin-induced testicular oxidative damage in rats. Food Chem Toxicol 2011;49:1115e21. [23] Masuda H, Tanaka T, Takahama U. Cisplatin generates superoxide anion by interaction with DNA in a cell-free system. Biochem Biophys Res Commun 1994;203:1175e80. [24] Ilbey YO, Ozbek E, Simsek A, Otunctemur A, Cekmen M, Somay A. Potential chemoprotective effect of melatonin in cyclophosphamide- and Cisplatin induced testicular damage in rats. Fertil Steril 2009;92:1124e32. [25] Mansilla S, Llovera L, Portugal J. Chemotherapeutic targeting of cell death pathways. Anticancer Agents Med Chem 2012;12:226e38. [26] Tang DG, La E, Kern J, Kehrer JP. Fatty acid oxidation and signaling in apoptosis. Biol Chem 2002;383:425e42. [27] Lirdi LC, Stumpp T, Sasso-Cerri E, Miraglia SM. Amifostine protective effect on cisplatin-treated rat testis. Anat Rec 2008;291:797e808. [28] Pectasides D, Pectasides M, Farmakis D, Nikolaou M, Koumpou M, Kostopoulou V, et al. Testicular function in patients with testicular cancer treated with Bleomycine EtoposideeCarboplatin (BEC90) combination chemotherapy. Eur Urol 2004;45:187e93. [29] Ilbey YO, Ozbek E, Simsek A, Cekmen M, Otunctemur A, Somay A. Chemoprotective Effect of a Nuclear Factor-kB Inhibitor, Pyrrolidine Dithiocarbamate, Against CisplatinInduced Testicular Damage in Rats. J Androl 2009;30: 505e14. [30] Aitken RJ, Curry BJ. Redox regulation of human sperm function: from the physiological control of sperm capacitation to the etiology of infertility and DNA damage in the germ line. Antioxid Redox Signal 2011;14:367e81.