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Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats
GENE-40264; No. of pages: 8; 4C: Gene xxx (2015) xxx–xxx
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Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats Rasha H. Mohamed a,⁎, Rehab A. Karam b, Hoda A. Hagrass c, Mona G. Amer d, Manal R. Abd El-Haleem d a
Biochemistry Department, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt Clinical Pathology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt d Histology and Cell Biology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt b c
a r t i c l e
i n f o
Article history: Received 31 October 2014 Received in revised form 5 February 2015 Accepted 9 February 2015 Available online xxxx Keywords: Doxorubicin SSCs Testicular toxicity Real-time PCR
a b s t r a c t The present study was designed to investigate whether spermatogonial stem cells (SSCs) have possible effect on doxorubicin (DOX)-induced testicular apoptosis and damaged oxidant/antioxidant balance in rats. Sixty male Albino rats were divided into 3 groups: the saline control group, the testicular toxicity group (2 mg/kg DOX once a week for 8 weeks) and the third group is a donor stem cells transplanted following pre-treatment with DOX. After the 8th week, the rats were sacrificed and tissues were collected and examined for CD95, CD95L, Caspase 3, and Caspase 8 gene expression using RT-PCR. While malondialdehyde (MDA), glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD) were determined using colorimetric kits. Biochemical, histopathological and PCR results showed improvement of the SSCs' group compared to the DOX-group. It was observed that spermatogonial stem cell affected DOX-induced activation of intrinsic apoptotic signaling pathway via preventing DOXinduced increases in CD95 and CD95L levels as well as cleaved Caspase-8 and Caspase-3 levels in testicular tissues, however, spermatogonial stem cell decreased Dox-induced NF-κB activation as well. It can be concluded that SSCs may be utilized to develop new cell-based therapies, and to advance germline gene therapy. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Each year, an estimated 160,000 children are diagnosed with cancer worldwide, and it has been estimated that a male infant has a 1 in 300 chance of being diagnosed with a malignancy by the age of 20 (Ries et al., 2007). Fortunately, success rates in treating childhood cancer have increased dramatically over the past few decades, and now approximately 80% of children survive following treatment (Steliarova-Foucher et al., 2004; Howlader et al., 2011). Many therapies to treat cancer are gonadotoxic and can lead to infertility, and fertility potential has an important impact on quality of life according to cancer survivors (Carter et al., 2005; Lee et al., 2006). In fact, the American Society of Clinical Oncology now recommends that the reproductive risks of cancer therapies and fertility preservation options should be routinely discussed with patients' prepubertal boys, adolescents, and adult men' before beginning treatment (Lee et al., 2006; Keros et al., 2007). Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout their lives, and Abbreviations: ANOVA, analysis of variance; CAT, catalase; DOX, doxorubicin; DMEM, Dulbecco's modified Eagle's medium; ELISA, high-sensitivity enzyme-linked immunosorbent assay; EDTA, ethylenediaminetetraacetic acid; GSH-Px, glutathione peroxidase; H2O2, hydrogen peroxide; MDA, malondialdehyde; NBT, nitroblue tetrazolium; NF-κB, nuclear factor kappa B; qPCR, quantitative real-time PCR; Cq, quantification cycle; St, seminiferous tubules; SOD, superoxide dismutase; SSCs, spermatogonial stem cells; TBA, thiobarbituric acid ⁎ Corresponding author at: 28 Elglaa Street, Zagazig, Egypt. E-mail address: [email protected] (R.H. Mohamed).
they are defined by their ability to undergo both self-renewing cell divisions and differentiation, leading to the production of haploid sperm. Regeneration of spermatogenesis following SSC transplantation has now been established in several animal models, including rodents, goats, sheep, pigs, dogs, and monkeys (Mikkola et al., 2006; Herrid et al., 2009; Hermann et al., 2012). Anthracycline antibiotics are widely used as chemotherapeutic drugs in the treatment of human hematological malignancies and solid tumors. Doxorubicin (DOX) has become one of the most prescribed anticancer drugs (Hannun, 1997). The clinical use of DOX is associated with testicular dysfunction characterized by altered sperm development, production, structural integrity and motility rates in association with increased cellular apoptosis (Kato et al., 2001; Prahalathan et al., 2005; Trivedi et al., 2011). The present work aims to investigate whether spermatogonial stem cells (SSCs) has possible effect against DOX-induced testicular apoptosis and damaged oxidant/antioxidant balance in rats. 2. Materials and methods 2.1. Drugs and chemicals Doxorubicin hydrochloride (50 mg) was purchased from Pfizer Company, Egypt (Adriblastina®). All other chemicals were purchased from Sigma Chemicals Co., (St. Louis, MO, USA).
http://dx.doi.org/10.1016/j.gene.2015.02.015 0378-1119/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: Mohamed, R.H., et al., Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.02.015
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2.2. Experimental protocol 2.2.1. Donor cells preparation and transplantation analysis Cells for transplantation were obtained from the testes of male white Albino rat between 5 and 10 days by a two-step enzymatic digestion protocol (Brinster and Avarbock, 1994). In this procedure, the tunica albuginea was first removed or peeled from the testis, thereby exposing the seminiferous tubules. The testes were then incubated in approximately 10 volumes of Hanks' balanced salt solution without calcium or magnesium (HBSS) containing 10 mg/ml collagenase (Type IV, Sigma) at 37 °C with gentle agitation for 15 min or until the tubules were separated. Dispersion of the tubules can be hastened by careful dissection, spreading of the tubules, and removal of intertubular cellular strands when visible. The addition of 300 μg/ml DNAse facilitates intertubular cell dispersion. The testis tubules were then washed 2 to 4 times in 3–4 ml of HBSS followed by incubation at 37 °C for 5 min in HBSS containing 1 mM EDTA and 0.25% trypsin. Separation and dispersion of the tubule cells can be hastened by pipetting and gentle agitation. When most of the cells were dispersed, the action of trypsin was terminated by adding a 10 to 20% volume of fetal bovine serum. The inclusion of 400 μg/ml of DNAse decreases stickiness and facilitates dispersion of the cells. Following digestion, any large pieces of undigested material were removed, and the cell suspension was filtered through a nylon mesh with 60 μm pore size to remove large clumps of cells. The filtrate was centrifuged at 600 ×g for 5 min at 16 °C and the supernatant was carefully removed from the pellet. The cells in the pellet were then resuspended in 400 μl Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum at a concentration of (2–3) × 107 cells/ml. The cells were maintained at 5 °C until the time of loading into an injection pipette, usually 1 to 4 h (Ogawa et al., 1997). 2.2.2. Transplantation of spermatogonial stem cells In order to inject donor cells directly into the rete testis, the testis was removed from the body cavity and reflected laterally. The rete lies primarily under the vascular pedicle where it contacts the surface. The most obvious vessels are the large veins that lie under the tunica and drain the testis. Insert the loaded micropipette into the area cranial to the vessels and adjacent to the efferent ducts. The micropipette should be almost parallel to the surface of the testis. After entering the rete, increase the pressure very slowly in the injection tube until the cell suspension fills the rete and flows into the tubules. About 10–20 μl of cell suspension was required to fill one testis. Filling of the tubules can be monitored by observing the movement of the cell suspension, which was facilitated by adding a small amount of trypan blue to the injection medium. After the tubules were completely filled, withdraw the micropipette. Return the testis to the abdomen and repeat the injection on the other testis. After replacing the testis in the abdominal cavity, suture the skin with one or two clips (Ogawa et al., 1997).
was injected with both DOX and spermatogonial stem cells, and in this group DOX was injected (i.p.) at a dose of 2 mg/kg body weight/week for 8 weeks while spermatogonial stem cells were administered as only a single dose after 4 weeks from DOX injections. Owing to the fact that rats need a period of 48–52 days for the exact spermatogenic cycle including spermatocytogenesis, meiosis and spermiogenesis (Türk et al., 2010; Çeribaşl et al., 2010), the administration period was set at 8 weeks. At the end of the experimental work, animals were sacrificed by decapitation. Blood samples were collected into tubes containing EDTA and centrifuged at 3000 rpm for 10 min to obtain plasma. One of the testes was prepared for light and electron microscope examination of seminiferous tubules (st). The other testes and plasma samples were stored at −20 °C until biochemical analyses. 2.4. Epididymal sperm concentration, motility, and abnormal sperm rate The epididymal sperm concentration in the right cauda epididymal tissue was determined with a hemocytometer using a modified method of Yokoi et al. (2003). Freshly isolated left cauda epididymal tissue was used for the analysis of sperm motility. The percentage sperm motility was evaluated using a light microscope with heated stage described by Sönmez et al. (2005). To determine the percentage of morphologically abnormal spermatozoa, the slides stained with eosin–nigrosin (1.67% eosin, 10% nigrosin and 0.1 M sodium citrate) were prepared. The slides were then viewed under a light microscope at 400× magnification. A total of 300 spermatozoa were examined on each slide, whereas the head, tail and total abnormality rates of spermatozoa were expressed as percentage (Ateşşahin et al., 2006). 2.5. Biochemical analysis 2.5.1. Preparation of tissue homogenates Tissue samples were homogenized in phosphate buffer (0.5 M, pH 7.0) (1/10, w/v) and then centrifuged for 5 min at 16,000 ×g at 4 °C to sediment unbroken cells and cellular debris. In the supernatant, the determination of lipid peroxidation, glutathione peroxidase activities, catalase, as well as superoxide dismutase content was determined. 2.5.2. Assay of oxidative stress 2.5.2.1. Lipid peroxidation. Lipid peroxide was estimated in testis homogenate by measurement of malondialdehyde (MDA) levels spectrophotometrically whereas the testis samples were homogenized in an ice-cold 50 mM potassium phosphate buffer (pH 7.5), centrifuged for 15 min at 12,000 ×g at 4 °C and then the supernatant was collected. MDA in the supernatant can react with freshly prepared thiobarbituric acid (TBA) to form a colored complex which has maximum absorbance at 535 nm. The nmol MDA/g wet tissue was calculated from the plotted standard curve prepared from 1,1,3,3-tetraethoxypropane (Buege and Aust, 1978).
2.3. Animals and experimental design 2.5.3. Assay of antioxidant enzymes The recipient rats were 15–30 days of age. The animals were maintained under standard conditions of humidity, temperature (25 ± 2 °C) and light (12 h light/12 h dark). They were fed with a standard rat diet and had free access to water. The study was conducted in accordance with the Guidelines for Ethical Care of Experimental Animals and was approved by the Animal Care and Use Committee of Biochemistry Department, Faculty of Pharmacy, Zagazig University. The animals were randomly divided into three groups, each of 20 rats. The first group was administered sterile saline and left as a control group. The second group was injected intraperitoneally (i.p.) with DOX; which already reconstituted in sterile saline; as a weekly single dose of 2 mg/kg body weight for 8 weeks ( eribaşl et al., 2012). The third group
2.5.3.1. Glutathione peroxidase (GSH-Px) activity. Glutathione peroxidase (GSH-Px) activity was spectrophotometrically determined according to the method of Lawrence and Burk (1976). The reaction mixture contains 1 ml of sodium phosphate buffer (pH 7.0) and 0.1 ml of NADPH reagent. Homogenate (0.01 ml) was added to the reaction mixture and then it was initiated by adding 0.1 ml of H2O2. The decrease in absorbance at 340 nm was recorded over a period of 3 min. Protein concentrations were determined using the method of Lowry et al. (1951). The GSH-Px activity was expressed as IU/g protein. 2.5.3.2. Catalase (CAT) activity. The catalase (CAT) activity was spectrophotometrically determined by measuring the decomposition of
Please cite this article as: Mohamed, R.H., et al., Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.02.015
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hydrogen peroxide (H2O2) in a phosphate buffer (pH 7.0) at 240 nm. The enzyme activity [I.U.] was defined as 1 μmol of H2O2 decomposed per minute. The final activity was expressed in units per gram of total soluble proteins (Aebi, 1984). 2.5.3.3. Superoxide dismutase (SOD) activity. The superoxide dismutase (SOD) activity was spectrophotometrically measured using xanthine and xanthine oxidases to generate superoxide radicals which react with nitroblue tetrazolium (NBT) and expressed as U/g tissue (Sun et al., 1988). 2.5.4. Determination of testosterone levels The plasma testosterone level was measured by ELISA method using a calbiotech ELISA testosterone kit according to the kit manufacturer's instructions and expressed as ng/ml. 2.5.5. Real-time quantitative analyses for CD95, CD95L, Caspase 3 and Caspase 8 gene expression Total RNA was extracted from testis tissue homogenate using RNeasy purification reagent (Qiagen, Valencia, CA). cDNA was generated from 5 μg of total RNA extracted with 1 μl (20 pmol) antisense primer and 0.8 μl superscript AMV reverse transcriptase for 60 min at 37 °C. The relative abundance of mRNA species was assessed using the SYBR Green method on an ABI prism 7500 sequence detector system (Applied Biosystems, Foster City, CA). The sequences of the oligonucleotide primers used in these assays were recorded in Table 1. All primer sets had a calculated annealing temperature of 60 °C. Quantitative realtime PCR (qPCR) was performed in duplicate in a 25 μl reaction volume consisting of 2 × SYBR Green PCR Master Mix (Applied Biosystems), 900 nM of each primer and 2–3 μl of cDNA. The PCR protocol included one cycle at 50 °C for 2 min, one cycle at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 10 min. Data from real-time assays were calculated using PE 7900 Sequence Detection System Software (Version 1.7; Applied Biosystems). Beta-actin was used for template normalizations. Fluorescent signals were normalized to an internal reference, and the quantification cycle (Cq) was set within the exponential phase of the PCR. The relative gene expression was calculated by comparing cycle times for each target PCR. Subtracting the β-actin Cq value, which provided the ΔCq value, normalized the target PCR Cq values. The relative expression level
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between treatments was then calculated using the following equation: relative gene expression change (R) = 2−(ΔCq sample − ΔCq control). 2.6. Histopathologic examinations One testis was quickly dissected and prepared for light and electron microscope examination of seminiferous tubules (st). Specimens were fixed in 10% buffered formalin and processed to prepare paraffin blocks, and serial sections of 5 μm were obtained and stained using methods described by Bancroft and Gamble (2002) for H&E stains and immunohistochemical staining for detection of nuclear factor kappa B (NF-κB) as a marker of apoptosis. Immunohistochemical reaction was carried out according to Kiernan (2000) with the use of an avidin biotin peroxidase system produced by Nova Castra Laboratories Ltd, Tyne, United Kingdom. The primary antibody used was NF kappa B/p65 (Rel A) Ab1 Rabbit Polyclonal Antibody (Neomarkers, Cat. #AP-9003, Labvision, Fremont, CA, USA). Positive control tissue was the placenta. For negative control, the primary antibody was replaced by a phosphate buffer solution. Finally, the NF-κB cytoplasmic and nuclear site of reaction was stained brown (Pentikainen et al., 2002). For transmission electron microscope examination, specimens were immediately fixed in 2.5% glutaraldehyde buffered with 0.1 mol/l phosphate buffer at a pH of 7.4 for 2 h and then post fixed in 1% osmium tetroxide in the same buffer for 1 h. They were processed to prepare stained ultrathin sections according to Glauert and Lewis (1998). 2.6.1. Quantitative morphometric measurements Serial sections stained with H&E and immunohistochemical reaction were morphometrically analyzed with a Leica Quin500C image analyzer computer system to evaluate the epithelial height of seminiferous tubules and area percentage of NF-κB immune reactions. Ten various fields were chosen from each slide. 2.7. Statistical analyses Data were presented as mean ± SD. Differences between groups were assessed by one-way analysis of variance (ANOVA) followed by post hoc multiple comparison tests (least significant different test; LSD) to compare changes among individual groups. The SPSS program (Version 10.0; SPSS, Chicago, IL) was used for the statistical analyses. The significance level was established at P b 0.05.
Table 1 Primer sequences used for quantitative real-time PCR reactions.
CD95
CD95 ligand
Caspase 8
Caspase 3
β-Actin
Primer sequences
Annealing temperature
Base pair product
Forward primer: 5-CGT GCA CAG AAGGGG AGG AG-3 Reverse primer: 5-ACATTCATTGGCACACTTTCA GGA-3 Forward primer: 5-ACTACCACCGCC TTC CCAACC-3 Reverse primer: 5-GGC CGC CTT TCT TAT ACT TCACTC-3 Forward primer: 5-CCCCACCCTCACTTTGCT-3 Reverse primer: 5-GGAGGACCAGGCACTTA-3 Forward primer: 5-CAGAGCTGGACTGCGGTATTGA-3 Reverse primer: 5-AGCATGGCGCAAAGTGACTG-3 Forward primer: 5-GAG AGG GAA ATC GTG CGT GAC-3 Reverse primer: 5-CAT CTG CTG GAA GGT GGA CA-3
56 °C
399
59 °C
368
60 °C
303
60 °C
320
55 °C
453
Please cite this article as: Mohamed, R.H., et al., Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.02.015
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3. Results 3.1. Sperm characteristics Epididymal sperm concentration, sperm motility, and abnormal sperm rates were presented at Table 2. DOX treatment caused significant (P b 0.01) decreases in sperm concentration and motility; it increased significantly the head, tail and total abnormality rates of sperm (P b 0.001) in comparison with the control group. Administration of SSCs with DOX caused a significant increase in the values of sperm concentration and motility (P b 0.001) in comparison with the DOX group. However, it caused a significant decrease in abnormal sperm rates (P b 0.001) as compared to the untreated group. 3.2. Biochemical parameters Testicular tissue lipid peroxidation levels, antioxidant enzyme activities and plasma testosterone levels are presented in Table 3. MDA and testosterone levels were significantly increased in the DOX group as compared to the control group (P ≤ 0.0001). The stem cell received group had lower levels of MDA and testosterone than the doxorubicin group (P ≤ 0.0001). A significant (P ≤ 0.0001) decrease in GSH-Px, CAT and SOD levels was observed in the DOX group in comparison with the control group. However, SSCs' administration to DOX-treated rats provided a significant (P ≤ 0.0001) increase in GSH-Px, CAT and SOD activities when compared with the values of the doxorubicin group. 3.3. Expression of CD95, CD95L, Caspase 8 and Caspase 3 genes Concerning gene expression, there was a significant increase in the CD95, CD95L, Caspase 8 and Caspase 3 genes of the doxorubicin group compared to normal one (P ≤ 0.0001), however, there was a significant decrease in these genes of the SSCs' treated group compared to the DOX group (P ≤ 0.0001) (Table 4).
in the basal cells of seminiferous tubules of the DOX-treated group (Fig. 2b) while no reaction was detected in the control group (Fig. 2a). Moreover, weak cytoplasmic reaction was observed in some cells lining seminiferous tubules of the stem cell received group (Fig. 2c). These results were confirmed by morphometrical analysis of area % of NF-κB immunoreactions (Table 5). Statistical analysis of the epithelial height of the seminiferous tubules of the DOX-treated group showed a highly significant decrease as compared with other groups. However, the stem cell received group showed a highly significant increase in their epithelial height as compared with the doxorubicin treated group (Table 5). 3.6. Electron microscope examination of seminiferous tubules (st) It was observed in Fig. 3 that the control seminiferous tubules contain Sertoli cells that rest on regular basement membrane (Fig. 3a). These cells had large euchromatic nuclei. Spermatogonia, primary spermatocytes, spermatids and mature sperms were seen. Tight junctions between sertoli cells forming blood testis barrier are intact (Fig. 3b). Cut section in mature sperms showed normal appearance in different parts of sperms in the control group (Fig. 3c). Leydig cells are of normal structure (Fig. 3d). Seminiferous tubules of the DOX-treated group appeared degenerated. Apoptotic basal cells with dense nuclei, wide separations between germinal epithelium and separation of basal cells from basement membrane together with thickened folded tubular basement membrane were observed in most of tubules (Fig. 3e, f, g). Affected sperms appeared with a distorted axoneme and mitochondrial sheath (Fig. 3h). Leydig cells appeared vacuolated and with dense bodies and lipid droplets filling cytoplasm (Fig. 3i). The epithelial cells lining seminiferous tubules of stem cell recipient groups appeared normal with all types of germinal epithelium and normal blood testis barrier. Intratubular vacuolations were observed in some of the tubules (Fig. 3j, k). Normal cut sections of mature sperms and Leydig cells were detected (Fig. 3l, m). 4. Discussion
3.4. Histological examination of the testis Examination of H&E-stained sections (Fig. 1) of specimens removed from control (Fig. 1a, b) and stem cell recipient groups (Fig. 1g, h) showed closely packed and organized seminiferous tubules (st) with little interstitium. The tubules are lined with stratified germinal epithelium and Sertoli cells. All types of cells had normal cellular attachment. Tubular lumens are filled with sperms. In contrast, accentuated damage of the seminiferous epithelium with depletion and vacuolization of the germinal epithelium, as well as appearance of darkly stained apoptotic nuclei in basal spermatogonia and Sertoli cells. Moreover, multinucleated cells were revealed in some of the tubules and also detached germ cells in the tubular lumen were frequently observed in the DOX-treated group (Fig. 1c, d, e, f). 3.5. NF-κB immunostaining Fig. 2 showed that there is a strong cytoplasmic reaction in most of the cells lining seminiferous tubules (st) and positive nuclear reaction
Drugs used for chemotherapy are well-known to produce toxic sideeffects in multiple organ systems. The most commonly affected are those organs containing self-renewing cell populations, including the reproductive system (Tomao et al., 2006). Gonadal injury, which is commonly observed following this type of treatment, has been less extensively investigated than other adverse effects of these drugs, perhaps because no life-threatening symptoms arise from damage to the gonads. Testicular toxicity and infertility after chemotherapy in young men are a well-documented phenomenon (Oktay, 2005). Several studies suggest that germ cells are targets of doxorubicin toxicity in the developing testis (Ateşşahin et al., 2006; eribaşl et al., 2012). The data presented here indicates that a dose of DOX once a week during testicular development could result in a reduced adult SSC population potentially as a result of the loss of SSCs during testis development or accelerated irreversible differentiation of SSCs into spermatogonia committed for sperm production. The differentiation of spermatozoa is maintained through the function of Leydig cells which secrete testosterone. So that a significant decrease in sperm
Table 2 Epididymal sperm concentrations, sperm motility, and abnormal sperm rates in all groups. Epididymal sperm concentration (million g−1)
Normal (n = 19) DOX (n = 10) DOX + SSCs (n = 16)
310.8 ± 11.6 92.4 ± 8.2⁎ 304.2 ± 22.6#
Sperm motility (%)
70.5 ± 1.9 34.3 ± 2.4⁎ 60 ± 2.7#
Abnormal sperm rate (%) Head
Tail
Total
2.2 ± 0.19 45.1 ± 7.07⁎ 33.2 ± 5.32#
3.5 ± 0.22 18.9 ± 1.8⁎ 15.9 ± 1.5#
5.7 ± 0.41 64 ± 8.8⁎ 49.1 ± 6.8#
⁎ Significant from control, P b 0.001. # Significant from the DOX group, P b 0.001.
Please cite this article as: Mohamed, R.H., et al., Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.02.015
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Table 3 Oxidative stress biomarkers and plasma testosterone level in studied groups. MDA (nmol/g tissue) Normal (n = 19) DOX (n = 10) DOX + SSCs (n = 16)
9.2 ± 1.3
GSH-Px (IU/g protein)
CAT (IU/g tissue)
SOD (U/g tissue)
Testosterone (ng/ml)
16.3 ± 2.4
6.1 ± 0.4
17.1 ± 0.66
2.5 ± 0.1
1.7 ± 0.5⁎
7.5 ± 1.4⁎
0.1 ± 0.03⁎
15.8 ± 1.1⁎#
2.0 ± 0.32⁎#
24.2 ± 2.8⁎
6.5 ± 0.3⁎
10.2 ± 1.6#
15.7 ± 1.3#
5.3 ± 0.54⁎#
⁎ Significant from control, P ≤ 0.0001. # Significant from the DOX group, P ≤ 0.0001.
concentration, sperm motility, and testosterone levels, and a significant increase in abnormal sperm rates were observed in this group (Tables 2 & 3) together with degenerated STs and interstitial cells (Fig. 3). DOX administration resulted in excessive lipid peroxidation with concomitant decrease in glutathione peroxidase (GSH-Px), catalase, and superoxide dismutase in rat testes (Table 3) as previous studies reported (Prahalathan et al., 2004, 2005). It has been reported that doxorubicin interacts with mitochondrial NADH-dehydrogenase to facilitate the production of the doxorubicin semiquinone free radical intermediate, which in a subsequent reaction with molecular oxygen forms ROS (Mimnaugh et al., 1985). Doxorubicin induced an increase in reactive oxygen species (ROS) levels or its direct effects on Leydig cell causing its impairment leading to a decrease in testosterone production (Endo et al., 2003; Ateşşahin et al., 2006). In this study, it was observed that the reason of the DOXinduced decreased testosterone concentration was mainly due to the damages in Leydig cells (Fig. 3). Because spermatozoa plasma membranes contain large quantities of polyunsaturated fatty acids and their cytoplasm contains low concentrations of scavenging enzymes, they are particularly susceptible to the damage induced by excessive ROS (Aitken and McLaughlin, 2007). Thus, the increase of free radicals in cells can induce the lipid peroxidation by oxidative breakdown of polyunsaturated fatty acids in membranes of cells. Obviously, peroxidation of sperm lipids destroys the structure of lipid matrix in the membranes of spermatozoa, and it is associated with rapid loss of intracellular ATP leading to axonemal damage, decreased sperm viability and increased mid-piece morphological defects, and it even completely inhibits spermatogenes in extreme cases (Türk et al., 2010). It has been reported that DOX-induced direct DNA fragmentation (Suominen et al., 2003), chromosomal aberrations (Au and Hsu, 1980), and oxidative stress (Prahalathan et al., 2005; Ateşşahin et al., 2006) causing a decrease in sperm count and motility as well as an increase in dead and abnormal sperm rates. Previous studies demonstrated that anticancer drugs induce apoptosis by a mitochondrial pathway that is independent of death receptors (Engels et al., 2000). The reason for the controversial role of CD95 in drug-induced cell death is currently unclear. Therefore, we investigated CD95, CD95L, Caspase 8, and Caspase 3 expression levels in testicular tissue in order to determine the possible mechanism of DOX-induced testis injury.
Table 4 Effect of SSCs on the expression of CD95, CD95L, Caspase-8 and Caspase-3 in the studied groups.
Normal (n = 19) DOX (n = 10) DOX + SSCs (n = 16)
CD95
CD95L
Caspase-3
Caspase-8
2.1 ± 0.1
1.1 ± 0.04
1.4 ± 0.05
0.8 ± 0.03
8.7 ± 0.9⁎
3.6 ± 0.2⁎
3.7 ± 0.2⁎
2.7 ± 0.5⁎
2.8 ± 0.3#
1.9 ± 0.15⁎#
1.6 ± 0.3#
1.3 ± 0.1#
⁎ Significant from control, P ≤ 0.0001. # Significant from the DOX group, P ≤ 0.0001.
We found that overexpression of CD95, CD95L, Caspase 8, and Caspase 3 genes in testicular tissues was observed in the DOX group compared to normal one (Table 4). This data is in accordance with previous studies (Massart et al., 2004; Kalivendi et al., 2005). CD95 was isolated as a type I transmembranous glycoprotein and is known to be expressed abundantly and to mediate apoptosis in a variety of immune and non-immune tissues (Watanabe-Fukunaga et al., 1992). CD95L was discovered and identified as a type II transmembranous protein belonging to the TNF superfamily, and it very efficiently triggers apoptosis in various cell types by coupling with the cell surface receptor CD95 (Nagata and Golstein, 1995). The testis represents the major non-lymphoid source of CD95L in the body. Testis toxicity may therefore be through Sertoli cells, or “nurse cells”, which form the blood–testis barrier by virtue of tight junctions, maintain homeostasis, provide nourishment for the developing sperm cells, and protect germ cells from toxic injury. Degradation of the support capacity of Sertoli cells and increased expression of CD95L on Sertoli cells and CD95 on germ cells consequently cause robust apoptosis in germ cells (Richburg, 2000). Activation of receptor upon ligand binding leads to the formation of a death-inducing signaling complex (DISC) consisting of the adaptor protein Fas-associated death domain (FADD) and pro-Caspase-8. In this process, pro-Caspase-8 is self-cleaved, activated, and released into the cytoplasm and in turn activates the effector Caspase (Caspase 3) responsible for the proteolytic degradation of structural and signaling proteins that eventually results in cell death (Brunelle and Zhang, 2010). Another mechanism of cell death depends on activation of NF-κB, which is a major transcription factor known to regulate numerous apoptosis-related genes (Rasoulpour and Boekelheide, 2005). Generation of oxidative stress due to DOX treatment is responsible for the activation of a redox-sensitive transcription factor, NF-κB (Li and Karin, 1999). Under nonstimulating conditions, NF-κB is inhibited by specific inhibitors that masks its nuclear localization and causes its retention in the cytoplasm. Activation of NF-κB occurs when these inhibitors are phosphorylated and degraded by TNF-α or IL-1β, thus releasing NF-κB for transport to the nucleus (Senftleben and Karin, 2002). In the rat testis, the NF-κB proteins are present in Sertoli cell and spermatocyte which may play a role in the regulation of stage-specific gene expression during rat spermatogenesis (Delfino and Walker, 1998). In the present investigation, strong nuclear and cytoplasmic immunohistochemical expression of NF-κB was detected in the DOX treated group as compared to the control group (Fig. 2). Thus, it can be inferred that DOX-induced oxidative stress led to an increase in the expression of NF-κB, which in turn, resulted in an enhanced expression of various genes like Caspase-3 leading to apoptosis in the testicular cells. The current histopathological examination of testicular tissues revealed that the doxorubicin treated group showed an increase in disorganization, vacuolization in both SNTs and in the germinal epithelium. Some of the tubules were found to be atrophied and deprived of the cellular structure within, which indicated severe germ cell toxicity induced by DOX (Fig. 1). Similar impaired spermatogenesis has been
Please cite this article as: Mohamed, R.H., et al., Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.02.015
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Fig. 1. H&E stained section of testes of experimental groups. Control group (1a, b): showing regular seminiferous tubules (st), narrow intertubular space with interstitial cell nuclei (arrow head), tubules are lined by stratified germinal epithelium (g) with spermatogonia (1), spermatid (2), sperms (3) and Sertoli (4). The doxorubicin-treated rats (1c, d, e, f): showing distorted seminiferous tubules (st) with disorganized germinal epithelial layers, wide intertubular spaces (s) and vacuolated (v) seminiferous tubules are seen in (c, d). Irregular atrophied tubules with a depleted germinal epithelium and sloughing of some cells into the lumen (L) are seen in (d). Darkly stained nuclei (arrows) of the basal spermatogonia and Sertoli cells are also seen in most of the tubules. (1f), multinuclear giant cells (curved arrow) in the lumen of the tubules. Stem cell recipient group (1g, h): Normal seminiferous tubules of the stem cell recipient group. The adjacent tubules are lined with many layers of germinal epithelium with intercellular vacuolations (v). The tubular lumen is filled with sperm (sp).
reported in DOX administered rat testes (Yeh et al., 2009). Also, degenerated Sertoli cells with disrupted tight junctions suggest that DOX damages immature Sertoli cells. Disruption of Sertoli cell function by toxicants can reduce their capacity to nurse germ cells, leading to apoptosis of the latter (Richburg, 2000). In those patients whose anticancer therapy clinically predicts a complete depletion of SSCs, the outlined approaches of germ cell transplantation and testicular grafting might offer options for fertility preservation. SSCs and their ability to self-renew are critical for maintenance of male fertility by supplying differentiating cells for sperm production in mammals. A technique for transplanting SSCs was first
described by Brinster and colleagues (Brinster and Zimmermann, 1994; Brinster and Avarbock, 1994), and it has become the “gold standard” for assessing stem cell activity in rodent testes. Restoration of fertility following SSCs' transplantation in rodents is suggesting therapeutic potential for the technique in humans. Here, we are showing that SSCs' injection to DOX rat resulted in a rapid reversal of tissue damage. Restoration of normal histological structure of testis was evident with the appearance of all germ cells including mature sperms with normal structure (Fig. 1). A previous study found that the donor germ cell suspension remains widely dispersed within the recipient seminiferous tubules during the
Fig. 2. Immunohistochemical reaction of NF-kB in seminiferous tubules showing negative reaction in the control group (a), strong nuclear reaction (arrow head) and cytoplasmic reaction in Sertoli cell (curved arrow) is seen in the adriamycin treated group (b). Weak positive immune reaction is seen in few cells of some SNTs (arrow) of the stem cell received group (c).
Please cite this article as: Mohamed, R.H., et al., Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.02.015
R.H. Mohamed et al. / Gene xxx (2015) xxx–xxx Table 5 Epithelial heights in seminiferous tubules and immunohistochemical reaction of NF-κB of studied groups.
Control DOX DOX + SSCs
Epithelial heights
Area % NF-κB
83.4 ± 0.5 70.8 ± 0.29⁎ 82.1 ± 3.9#
2.97 ± 0.25 8.2 ± 0.56⁎ 1.37 ± 0.10#
⁎ Significant from control, P ≤ 0.0001. # Significant from the DOX group, P ≤ 0.0001.
first weeks following transplantation. At 4 week post transplantation, donor germ cells are divided on the basement membrane of the recipient seminiferous tubuli, forming a network. Differentiation of donor germ cells to more mature germ cell stages appears to begin after this initial colonization and spreading on the recipient seminiferous tubule basement membrane (Nagano et al., 1998).
7
In the present work, SSCs' injection to DOX rat caused a diminished tissue level of CD95, CD95L, Caspase 8, Caspase 3 and immune expression of NF-κB, moreover, elevated production of antioxidant enzymes in DOX/SSCs' rat was observed. When SSCs were harvested from donor testes and transplanted into a sterilized recipient testis, morphologically and functionally normal spermatogenesis was reestablished. These results are consistent with previous findings by others (Lo et al., 2005; Becker and Jakse, 2007) that revealed a successful transplantation of SSCs and that Leydig cell progenitors conserve the fertility and antiapoptotic effect of SSCs. Our data revealed that SSCs can be a potent candidate therapeutic treatment for infertility. 5. Conclusion In the present study, we observed that spermatogonial stem cell prevented DOX-induced activation of intrinsic apoptotic signaling
Fig. 3. Electron micrograph of seminiferous tubules showing the control group (3a, b, c, d): Sertoli cell (S) resting on thin regular basement membrane (B), spermatogonia (sg), 1ry spermatocytes and spermatids (st) and normal tight junction between Sertoli cells (double arrows). (3c): Cut section of the mature sperms (arrows) appears normal. Observe the axoneme, mitochondrial sheath and fibrous sheath. (3d): Leydig cells with large irregular nucleus and mitochondria with tubular cristae (curved arrows). (3e, f, g, h, i): Seminiferous tubules of DOXtreated group, germ cells (g) are widely separated and large heterogeneous bodies (H) are seen. Spermatogonia (sg) are separated (stars) from underlying basement membrane (B). (3f): Sertoli cell nucleus (Sn) is deformed (arrow head) and its cytoplasm contains vacuoles (v). (3g): Apoptotic basal cell with dense nucleus (d), disrupted cellular junctions (star) and thickened infolded tubular basement membrane (B). (3h): Affected sperms (arrows) appear with distorted axoneme and mitochondrial sheath. (3i): Leydig cells' cytoplasm appears vacuolated (V) and contains dense bodies (d) and lipid droplets (L). Seminiferous tubules of stem cell recipient group (3j, k, l, m): appears normal with Sertoli cell (S), primary spermatocyte (1S) resting on thin regular basement membrane (B) and normal tight junction between Sertoli and other germ cells (double arrows). Normal organized cut section of sperms is seen in (3l). Leydig cell is also normal with some vacuoles (3m).
Please cite this article as: Mohamed, R.H., et al., Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.02.015
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pathway. Specifically, SSCs prevented DOX-induced increases in CD95 and CD95L levels and cleaved Caspase-8 and Caspase-3 levels in testicular tissues. In addition, spermatogonial stem cell decreased Doxinduced nuclear factor-kappa B (NF-κB) activation. Finally, it should be noted that SSCs may also offer a renewable source of cells to be used to correct many diseases of aging, to develop new cell-based therapies, and to advance germline gene therapy. Conflict of interest The authors declare no conflict of interest. Acknowledgments This study was funded with the support of academic research in Zagazig University Projects, Zagazig University Post Graduate & Research Affairs (4k1/M5). References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Aitken, R.J., McLaughlin, E.A., 2007. Molecular mechanisms of sperm capacitation: progesterone-induced secondary calcium oscillations reflect the attainment of a capacitated state. Soc. Reprod. Fertil. Suppl. 63, 273–293. Ateşşahin, A., Karahan, I., Türk, G., Gür, S., Yılmaz, S., Çeribas, A.O., 2006. Protective role of lycopene on cisplatin induced changes in sperm characteristics, testicular damage and oxidative stress in rats. Reprod. Toxicol. 21, 42–47. Au, W.W., Hsu, T.C., 1980. The genotoxic effects of adriamycin in somatic and germinal cells of the mouse. Mutat. Res. 79, 351–361. Bancroft, J., Gamble, A., 2002. Theory and Practice of Histological Techniques. 5th ed. Churchill Livingstone, New York, London, pp. 165–180. Becker, C., Jakse, G., 2007. Stem cells for regeneration of urological structures. Eur. Urol. 51, 1217–1228. Brinster, R.L., Avarbock, M.R., 1994. Germline transmission of donor haplotype following spermatogonial transplantation. Proc. Natl. Acad. Sci. 91, 11303–11307. Brinster, R.L., Zimmermann, J.W., 1994. Spermatogenesis following male germ-cell transplantation. Proc. Natl. Acad. Sci. 91, 11298–11302. Brunelle, J.K., Zhang, B., 2010. Apoptosis assays for quantifying the bioactivity of anticancer drug products. Drug Resist. Updat. 13, 172–179. Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. Methods Enzymol. 52, 302–310. Carter, J., Rowland, K., Chi, D., Brown, C., Abu-Rustum, N., Castiel, M., 2005. Gynecologic cancer treatment and the impact of cancer-related infertility. Gynecol. Oncol. 97, 90–95. eribaşl, A.O., Sakin, F., Türk, G., Sönmez, M., Ateşşahin, A., 2012. Impact of ellagic acid on adriamycin-induced testicular histopathological lesions, apoptosis, lipid peroxidation and sperm damages. Exp. Toxicol. Pathol. 64, 717–724. Çeribaşl, A.O., Türk, G., Sönmez, M., Sakin, F., Ateşşahin, A., 2010. Toxic effect of cyclophosphamide on sperm morphology, testicular histology and blood oxidant–antioxidant balance, and protective roles of lycopene and ellagic acid. Basic Clin. Pharmacol. Toxicol. 107, 730–736. Delfino, F., Walker, W.H., 1998. Stage-specific nuclear expression of NF-κB in mammalian testis. Mol. Endocrinol. 12, 1696–1707. Endo, F., Manabe, F., Takeshima, H., Akaza, H., 2003. Protecting spermatogonia from apoptosis induced by doxorubicine using the luteinizing hormone-releasing hormone analog leuprorelin. Int. J. Urol. 10, 72–77. Engels, I.H., Stepczynska, A., Stroh, C., Lauber, K., Berg, C., Schwenzer, R., Wajant, H., Ja-Ènicke, R.U., Porter, A.G., Belka, C., Gregor, M., Schulze-Ostho, K., Wesselborg, S., 2000. Caspase-8/FLICE functions as an executioner caspase in anticancer druginduced apoptosis. Oncogene 19, 4563–4573. Glauert, A.M., Lewis, P.R., 1998. Biological Specimen Preparation for Transmission Electron Microscopy. 1st ed. Princeton University Press, London. Hannun, Y.A., 1997. Apoptosis and the dilemma of cancer chemotherapy. Blood 89, 1845–1853. Herrid, M., Olejnik, J., Jackson, M., Suchowerska, N., Stockwell, S., Davey, R., Hutton, K., Hope, S., Hill, J.R., 2009. Irradiation enhances the efficiency of testicular germ cell transplantation in sheep. Biol. Reprod. 81, 898–905. Hermann, B.P., Sukhwani, M., Winkler, F., Pascarella, J.N., Peters, K.A., Sheng, Y., Valli, H., Rodriguez, M., Ezzelarab, M., Dargo, G., Peterson, K., Masterson, K., Ramsey, C., Ward, T., Lienesch, M., Volk, A., Cooper, D.K., Thomson, A.W., Kiss, J.E., Penedo, M.T., Schatten, G.P., Mitalipov, S., Orwig, K.E., 2012. Spermatogonial stem cell transplantation into Rhesus testes regenerates spermatogenesis producing functional sperm. Cell Stem Cell 11, 715–726. Howlader, N., Noone, A.M., Krapcho, M., 2011. SEER Cancer Statistic Review 1975–2008. National Cancer Institute, Bethesda, Maryland, USA. Kato, M., Makino, S., Kimura, H., Ota, T., Furuhashi, T., Nagamura, Y., 2001. Sperm motion analysis in rats treated with adriamycin and its applicability to male reproductive toxicity studies. J. Toxicol. Sci. 26, 51–59.
Kalivendi, S.V., Konorev, E.A., Cunningham, S., Vanamala, S.K., Kaji, E.H., Joseph, J., Kalyanaraman, B., 2005. Doxorubicin activates nuclear factor of activated Tlymphocytes and Fas ligand transcription: role of mitochondrial reactive oxygen species and calcium. Biochem. J. 389, 527–539. Keros, V., Hultenby, K., Borgström, B., Fridström, M., Jahnukainen, K., Hovatta, O., 2007. Methods of cryopreservation of testicular tissue with viable spermatogonia in pre-pubertal boys undergoing gonadotoxic cancer treatment. Hum. Reprod. 22, 1384–1395. Kiernan, J.A., 2000. Histological and histochemical methods. Theory and Practice, 3rd ed. Butterworth Hememann/Johanne Shurp, Oxford, Auckland, Boston/Melbourne, New Delln, pp. 267–281 (390–418). Lawrence, R.A., Burk, R.F., 1976. Glutathione peroxidase activity in selenium-deficient rat liver. Biochem. Biophys. Res. Commun. 71, 952–958. Lee, S.J., Schover, L.R., Partridge, A.H., Patrizio, P., Wallace, W.H., Hagerty, K., Beck, L.N., Brennan, L.V., Oktay, K., 2006. American Society of Clinical Oncology recommendations on fertility preservation in cancer patients. J. Clin. Oncol. 24, 2917–2931. Li, N., Karin, M., 1999. Is NF-kappa B the sensor of oxidative stress? FASEB J. 13, 1137–1143. Lo, K.C., Whirledge, S., Lamb, D.J., 2005. Stem cells: implications for urology. Curr. Urol. Rep. 6, 49–54. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 193, 265–275. Massart, C., Barbet, R., Genetet, N., Gibassier, J., 2004. Doxorubicin induces Fas-mediated apoptosis in human thyroid carcinoma cells. Thyroid 14, 263–270. Mikkola, M., Sironen, A., Kopp, C., Taponen, J., Sukura, A., Vilkki, J., Katila, T., Andersson, M., 2006. Transplantation of normal boar testicular cells resulted in complete focal spermatogenesis in a boar affected by the immotile short-tail sperm defect. Reprod. Domest. Anim. 41, 124–128. Mimnaugh, E.G., Trush, M.A., Bhatnagar, M., Gram, T.E., 1985. Enhancement of reactive oxygen-dependent mitochondrial membrane lipid peroxidation by the anticancer drug adriamycin. Biochem. Pharmacol. 34, 847–856. Nagata, S., Golstein, P., 1995. The Fas death factor. Science 267, 1449–1456. Nagano, M., Avarbock, M.R., Leonida, E.B., Brinster, C.J., Brinster, R.L., 1998. Culture of mouse spermatogonial stem cells. Tissue Cell 30, 389–397. Ogawa, T., Arechaga, J.M., Avarboc, M.R., Brinster, R.L., 1997. Transplantation of testis germinal cells into mouse seminiferous tubules. Int. J. Dev. Biol. 41, 111–122. Oktay, K., 2005. Fertility preservation: an emerging discipline in the care of young patients with cancer. Lancet Oncol. 6, 192–193. Pentikainen, V., Suomalainen, L., Erkkila, K., Martelin, E., Parvinen, M., Markku, O., Dunkel, L., 2002. Nuclear factor-κB activation in human testicular apoptosis. Am. J. Pathol. 160, 205–218. Prahalathan, C., Selvakumar, E., Varalakshmi, P., 2004. Remedial effect of DL-α-lipoic acid against adriamycin induced testicular lipid peroxidation. Mol. Cell. Biochem. 267, 209–214. Prahalathan, C., Selvakumar, E., Varalakshmi, P., 2005. Protective effect of lipoic acid on adriamycin-induced testicular toxicity. Clin. Chim. Acta 360, 160–166. Rasoulpour, R.J., Boekelheide, K., 2005. NF-κB is activated in the rat testis following exposure to mono-(2-ethylhexyl) phthalate. Biol. Reprod. 72, 479–486. Richburg, J.H., 2000. The relevance of spontaneous-and chemically-induced alterations in testicular germ cell apoptosis to toxicology. Toxicol. Lett. 15, 79–86. Ries, L.A.G., Melbert, D., Krapcho, M., Mariotto, A., Miller, B.A., Feuer, E.J., Clegg, L., Horner, M.J., Howlader, N., Eisner, M.P., Reichman, M., Edwards, B.K., 2007. SEER Cancer Statistics Review, 1975–2004. National Cancer Institute, Bethesda, Maryland, USA. Senftleben, U., Karin, M., 2002. The IKK/NF-kappa B pathway. Crit. Care Med. 30, S18–S26. Sönmez, M., Türk, G., Yüce, A., 2005. The effect of ascorbic acid supplementation on sperm quality, lipid peroxidation and testosterone levels of male Wistar rats. Theriogenology 63, 2063–2072. Steliarova-Foucher, E., Stiller, C., Kaatsch, P., Berrino, F., Coebergh, J.W., Lacour, B., Parkin, M., 2004. Geographical patterns and time trends of cancer incidence and survival among children and adolescents in Europe since the 1970s (the ACCIS project): an epidemiological study. Lancet 364, 2097–2105. Suominen, J.S., Linderborg, J., Nikula, H., Hakovirta, H., Parvinen, M., Toppari, J., 2003. The effects of mono-2-ethylhexyl phathalate, adriamycin and N-ethyl-N-nitrosourea onstage-specific apoptosis and DNA synthesis in the mouse spermatogenesis. Toxicol. Lett. 143, 163–173. Sun, Y., Oberley, L.W., Li, Y., 1988. A simple method for clinical assay of superoxide dismutase. Clin. Chem. 34, 497–500. Tomao, F., Miele, E., Spinelli, G.P., Tomao, S., 2006. Anticancer treatment and fertility effects. Literature review. J. Exp. Clin. Cancer Res. 25, 475–481. Trivedi, P.P., Tripathi, D.N., Jena, G.B., 2011. Hesperetin protects testicular toxicity of doxorubicin in rat: role of NF kappa B, p38 and caspase-3. Food Chem. Toxicol. 49, 838–847. Türk, G., Çeribaş, A.O., Sakin, F., Sönmez, M., Ateşşahin, A., 2010. Antiperoxidative and anti-apoptotic effects of lycopene and ellagic acid on cyclophosphamide-induced testicular lipid peroxidation and apoptosis. Reprod. Fertil. Dev. 22, 587–596. Watanabe-Fukunaga, R., Brannan, C.I., Itoh, N., Yonehara, S., Copeland, N.G., Jenkins, N.A., Nagata, S., 1992. The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen. J. Immunol. 148, 1274–1279. Yeh, Y.C., Liu, T.J., Wang, L.C., Lee, H.W., Ting, C.T., Lee, W., Hung, C.J., Wang, K.Y., Lai, H.C., Lai, H.C., 2009. A standardized extract of Ginkgo biloba suppresses doxorubicininduced oxidative stress and p53-mediated mitochondrial apoptosis in rat testes. Br. J. Pharmacol. 156, 48–61. Yokoi, K., Uthus, E.O., Nielsen, F.H., 2003. Nickel deficiency diminishes sperm quantity and movement in rats. Biol. Trace Elem. Res. 93, 141–153.
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Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats Rasha H. Mohamed a,⁎, Rehab A. Karam b, Hoda A. Hagrass c, Mona G. Amer d, Manal R. Abd El-Haleem d a
Biochemistry Department, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt Clinical Pathology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt d Histology and Cell Biology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt b c
a r t i c l e
i n f o
Article history: Received 31 October 2014 Received in revised form 5 February 2015 Accepted 9 February 2015 Available online xxxx Keywords: Doxorubicin SSCs Testicular toxicity Real-time PCR
a b s t r a c t The present study was designed to investigate whether spermatogonial stem cells (SSCs) have possible effect on doxorubicin (DOX)-induced testicular apoptosis and damaged oxidant/antioxidant balance in rats. Sixty male Albino rats were divided into 3 groups: the saline control group, the testicular toxicity group (2 mg/kg DOX once a week for 8 weeks) and the third group is a donor stem cells transplanted following pre-treatment with DOX. After the 8th week, the rats were sacrificed and tissues were collected and examined for CD95, CD95L, Caspase 3, and Caspase 8 gene expression using RT-PCR. While malondialdehyde (MDA), glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD) were determined using colorimetric kits. Biochemical, histopathological and PCR results showed improvement of the SSCs' group compared to the DOX-group. It was observed that spermatogonial stem cell affected DOX-induced activation of intrinsic apoptotic signaling pathway via preventing DOXinduced increases in CD95 and CD95L levels as well as cleaved Caspase-8 and Caspase-3 levels in testicular tissues, however, spermatogonial stem cell decreased Dox-induced NF-κB activation as well. It can be concluded that SSCs may be utilized to develop new cell-based therapies, and to advance germline gene therapy. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Each year, an estimated 160,000 children are diagnosed with cancer worldwide, and it has been estimated that a male infant has a 1 in 300 chance of being diagnosed with a malignancy by the age of 20 (Ries et al., 2007). Fortunately, success rates in treating childhood cancer have increased dramatically over the past few decades, and now approximately 80% of children survive following treatment (Steliarova-Foucher et al., 2004; Howlader et al., 2011). Many therapies to treat cancer are gonadotoxic and can lead to infertility, and fertility potential has an important impact on quality of life according to cancer survivors (Carter et al., 2005; Lee et al., 2006). In fact, the American Society of Clinical Oncology now recommends that the reproductive risks of cancer therapies and fertility preservation options should be routinely discussed with patients' prepubertal boys, adolescents, and adult men' before beginning treatment (Lee et al., 2006; Keros et al., 2007). Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout their lives, and Abbreviations: ANOVA, analysis of variance; CAT, catalase; DOX, doxorubicin; DMEM, Dulbecco's modified Eagle's medium; ELISA, high-sensitivity enzyme-linked immunosorbent assay; EDTA, ethylenediaminetetraacetic acid; GSH-Px, glutathione peroxidase; H2O2, hydrogen peroxide; MDA, malondialdehyde; NBT, nitroblue tetrazolium; NF-κB, nuclear factor kappa B; qPCR, quantitative real-time PCR; Cq, quantification cycle; St, seminiferous tubules; SOD, superoxide dismutase; SSCs, spermatogonial stem cells; TBA, thiobarbituric acid ⁎ Corresponding author at: 28 Elglaa Street, Zagazig, Egypt. E-mail address: [email protected] (R.H. Mohamed).
they are defined by their ability to undergo both self-renewing cell divisions and differentiation, leading to the production of haploid sperm. Regeneration of spermatogenesis following SSC transplantation has now been established in several animal models, including rodents, goats, sheep, pigs, dogs, and monkeys (Mikkola et al., 2006; Herrid et al., 2009; Hermann et al., 2012). Anthracycline antibiotics are widely used as chemotherapeutic drugs in the treatment of human hematological malignancies and solid tumors. Doxorubicin (DOX) has become one of the most prescribed anticancer drugs (Hannun, 1997). The clinical use of DOX is associated with testicular dysfunction characterized by altered sperm development, production, structural integrity and motility rates in association with increased cellular apoptosis (Kato et al., 2001; Prahalathan et al., 2005; Trivedi et al., 2011). The present work aims to investigate whether spermatogonial stem cells (SSCs) has possible effect against DOX-induced testicular apoptosis and damaged oxidant/antioxidant balance in rats. 2. Materials and methods 2.1. Drugs and chemicals Doxorubicin hydrochloride (50 mg) was purchased from Pfizer Company, Egypt (Adriblastina®). All other chemicals were purchased from Sigma Chemicals Co., (St. Louis, MO, USA).
http://dx.doi.org/10.1016/j.gene.2015.02.015 0378-1119/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: Mohamed, R.H., et al., Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.02.015
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R.H. Mohamed et al. / Gene xxx (2015) xxx–xxx
2.2. Experimental protocol 2.2.1. Donor cells preparation and transplantation analysis Cells for transplantation were obtained from the testes of male white Albino rat between 5 and 10 days by a two-step enzymatic digestion protocol (Brinster and Avarbock, 1994). In this procedure, the tunica albuginea was first removed or peeled from the testis, thereby exposing the seminiferous tubules. The testes were then incubated in approximately 10 volumes of Hanks' balanced salt solution without calcium or magnesium (HBSS) containing 10 mg/ml collagenase (Type IV, Sigma) at 37 °C with gentle agitation for 15 min or until the tubules were separated. Dispersion of the tubules can be hastened by careful dissection, spreading of the tubules, and removal of intertubular cellular strands when visible. The addition of 300 μg/ml DNAse facilitates intertubular cell dispersion. The testis tubules were then washed 2 to 4 times in 3–4 ml of HBSS followed by incubation at 37 °C for 5 min in HBSS containing 1 mM EDTA and 0.25% trypsin. Separation and dispersion of the tubule cells can be hastened by pipetting and gentle agitation. When most of the cells were dispersed, the action of trypsin was terminated by adding a 10 to 20% volume of fetal bovine serum. The inclusion of 400 μg/ml of DNAse decreases stickiness and facilitates dispersion of the cells. Following digestion, any large pieces of undigested material were removed, and the cell suspension was filtered through a nylon mesh with 60 μm pore size to remove large clumps of cells. The filtrate was centrifuged at 600 ×g for 5 min at 16 °C and the supernatant was carefully removed from the pellet. The cells in the pellet were then resuspended in 400 μl Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum at a concentration of (2–3) × 107 cells/ml. The cells were maintained at 5 °C until the time of loading into an injection pipette, usually 1 to 4 h (Ogawa et al., 1997). 2.2.2. Transplantation of spermatogonial stem cells In order to inject donor cells directly into the rete testis, the testis was removed from the body cavity and reflected laterally. The rete lies primarily under the vascular pedicle where it contacts the surface. The most obvious vessels are the large veins that lie under the tunica and drain the testis. Insert the loaded micropipette into the area cranial to the vessels and adjacent to the efferent ducts. The micropipette should be almost parallel to the surface of the testis. After entering the rete, increase the pressure very slowly in the injection tube until the cell suspension fills the rete and flows into the tubules. About 10–20 μl of cell suspension was required to fill one testis. Filling of the tubules can be monitored by observing the movement of the cell suspension, which was facilitated by adding a small amount of trypan blue to the injection medium. After the tubules were completely filled, withdraw the micropipette. Return the testis to the abdomen and repeat the injection on the other testis. After replacing the testis in the abdominal cavity, suture the skin with one or two clips (Ogawa et al., 1997).
was injected with both DOX and spermatogonial stem cells, and in this group DOX was injected (i.p.) at a dose of 2 mg/kg body weight/week for 8 weeks while spermatogonial stem cells were administered as only a single dose after 4 weeks from DOX injections. Owing to the fact that rats need a period of 48–52 days for the exact spermatogenic cycle including spermatocytogenesis, meiosis and spermiogenesis (Türk et al., 2010; Çeribaşl et al., 2010), the administration period was set at 8 weeks. At the end of the experimental work, animals were sacrificed by decapitation. Blood samples were collected into tubes containing EDTA and centrifuged at 3000 rpm for 10 min to obtain plasma. One of the testes was prepared for light and electron microscope examination of seminiferous tubules (st). The other testes and plasma samples were stored at −20 °C until biochemical analyses. 2.4. Epididymal sperm concentration, motility, and abnormal sperm rate The epididymal sperm concentration in the right cauda epididymal tissue was determined with a hemocytometer using a modified method of Yokoi et al. (2003). Freshly isolated left cauda epididymal tissue was used for the analysis of sperm motility. The percentage sperm motility was evaluated using a light microscope with heated stage described by Sönmez et al. (2005). To determine the percentage of morphologically abnormal spermatozoa, the slides stained with eosin–nigrosin (1.67% eosin, 10% nigrosin and 0.1 M sodium citrate) were prepared. The slides were then viewed under a light microscope at 400× magnification. A total of 300 spermatozoa were examined on each slide, whereas the head, tail and total abnormality rates of spermatozoa were expressed as percentage (Ateşşahin et al., 2006). 2.5. Biochemical analysis 2.5.1. Preparation of tissue homogenates Tissue samples were homogenized in phosphate buffer (0.5 M, pH 7.0) (1/10, w/v) and then centrifuged for 5 min at 16,000 ×g at 4 °C to sediment unbroken cells and cellular debris. In the supernatant, the determination of lipid peroxidation, glutathione peroxidase activities, catalase, as well as superoxide dismutase content was determined. 2.5.2. Assay of oxidative stress 2.5.2.1. Lipid peroxidation. Lipid peroxide was estimated in testis homogenate by measurement of malondialdehyde (MDA) levels spectrophotometrically whereas the testis samples were homogenized in an ice-cold 50 mM potassium phosphate buffer (pH 7.5), centrifuged for 15 min at 12,000 ×g at 4 °C and then the supernatant was collected. MDA in the supernatant can react with freshly prepared thiobarbituric acid (TBA) to form a colored complex which has maximum absorbance at 535 nm. The nmol MDA/g wet tissue was calculated from the plotted standard curve prepared from 1,1,3,3-tetraethoxypropane (Buege and Aust, 1978).
2.3. Animals and experimental design 2.5.3. Assay of antioxidant enzymes The recipient rats were 15–30 days of age. The animals were maintained under standard conditions of humidity, temperature (25 ± 2 °C) and light (12 h light/12 h dark). They were fed with a standard rat diet and had free access to water. The study was conducted in accordance with the Guidelines for Ethical Care of Experimental Animals and was approved by the Animal Care and Use Committee of Biochemistry Department, Faculty of Pharmacy, Zagazig University. The animals were randomly divided into three groups, each of 20 rats. The first group was administered sterile saline and left as a control group. The second group was injected intraperitoneally (i.p.) with DOX; which already reconstituted in sterile saline; as a weekly single dose of 2 mg/kg body weight for 8 weeks ( eribaşl et al., 2012). The third group
2.5.3.1. Glutathione peroxidase (GSH-Px) activity. Glutathione peroxidase (GSH-Px) activity was spectrophotometrically determined according to the method of Lawrence and Burk (1976). The reaction mixture contains 1 ml of sodium phosphate buffer (pH 7.0) and 0.1 ml of NADPH reagent. Homogenate (0.01 ml) was added to the reaction mixture and then it was initiated by adding 0.1 ml of H2O2. The decrease in absorbance at 340 nm was recorded over a period of 3 min. Protein concentrations were determined using the method of Lowry et al. (1951). The GSH-Px activity was expressed as IU/g protein. 2.5.3.2. Catalase (CAT) activity. The catalase (CAT) activity was spectrophotometrically determined by measuring the decomposition of
Please cite this article as: Mohamed, R.H., et al., Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.02.015
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hydrogen peroxide (H2O2) in a phosphate buffer (pH 7.0) at 240 nm. The enzyme activity [I.U.] was defined as 1 μmol of H2O2 decomposed per minute. The final activity was expressed in units per gram of total soluble proteins (Aebi, 1984). 2.5.3.3. Superoxide dismutase (SOD) activity. The superoxide dismutase (SOD) activity was spectrophotometrically measured using xanthine and xanthine oxidases to generate superoxide radicals which react with nitroblue tetrazolium (NBT) and expressed as U/g tissue (Sun et al., 1988). 2.5.4. Determination of testosterone levels The plasma testosterone level was measured by ELISA method using a calbiotech ELISA testosterone kit according to the kit manufacturer's instructions and expressed as ng/ml. 2.5.5. Real-time quantitative analyses for CD95, CD95L, Caspase 3 and Caspase 8 gene expression Total RNA was extracted from testis tissue homogenate using RNeasy purification reagent (Qiagen, Valencia, CA). cDNA was generated from 5 μg of total RNA extracted with 1 μl (20 pmol) antisense primer and 0.8 μl superscript AMV reverse transcriptase for 60 min at 37 °C. The relative abundance of mRNA species was assessed using the SYBR Green method on an ABI prism 7500 sequence detector system (Applied Biosystems, Foster City, CA). The sequences of the oligonucleotide primers used in these assays were recorded in Table 1. All primer sets had a calculated annealing temperature of 60 °C. Quantitative realtime PCR (qPCR) was performed in duplicate in a 25 μl reaction volume consisting of 2 × SYBR Green PCR Master Mix (Applied Biosystems), 900 nM of each primer and 2–3 μl of cDNA. The PCR protocol included one cycle at 50 °C for 2 min, one cycle at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 10 min. Data from real-time assays were calculated using PE 7900 Sequence Detection System Software (Version 1.7; Applied Biosystems). Beta-actin was used for template normalizations. Fluorescent signals were normalized to an internal reference, and the quantification cycle (Cq) was set within the exponential phase of the PCR. The relative gene expression was calculated by comparing cycle times for each target PCR. Subtracting the β-actin Cq value, which provided the ΔCq value, normalized the target PCR Cq values. The relative expression level
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between treatments was then calculated using the following equation: relative gene expression change (R) = 2−(ΔCq sample − ΔCq control). 2.6. Histopathologic examinations One testis was quickly dissected and prepared for light and electron microscope examination of seminiferous tubules (st). Specimens were fixed in 10% buffered formalin and processed to prepare paraffin blocks, and serial sections of 5 μm were obtained and stained using methods described by Bancroft and Gamble (2002) for H&E stains and immunohistochemical staining for detection of nuclear factor kappa B (NF-κB) as a marker of apoptosis. Immunohistochemical reaction was carried out according to Kiernan (2000) with the use of an avidin biotin peroxidase system produced by Nova Castra Laboratories Ltd, Tyne, United Kingdom. The primary antibody used was NF kappa B/p65 (Rel A) Ab1 Rabbit Polyclonal Antibody (Neomarkers, Cat. #AP-9003, Labvision, Fremont, CA, USA). Positive control tissue was the placenta. For negative control, the primary antibody was replaced by a phosphate buffer solution. Finally, the NF-κB cytoplasmic and nuclear site of reaction was stained brown (Pentikainen et al., 2002). For transmission electron microscope examination, specimens were immediately fixed in 2.5% glutaraldehyde buffered with 0.1 mol/l phosphate buffer at a pH of 7.4 for 2 h and then post fixed in 1% osmium tetroxide in the same buffer for 1 h. They were processed to prepare stained ultrathin sections according to Glauert and Lewis (1998). 2.6.1. Quantitative morphometric measurements Serial sections stained with H&E and immunohistochemical reaction were morphometrically analyzed with a Leica Quin500C image analyzer computer system to evaluate the epithelial height of seminiferous tubules and area percentage of NF-κB immune reactions. Ten various fields were chosen from each slide. 2.7. Statistical analyses Data were presented as mean ± SD. Differences between groups were assessed by one-way analysis of variance (ANOVA) followed by post hoc multiple comparison tests (least significant different test; LSD) to compare changes among individual groups. The SPSS program (Version 10.0; SPSS, Chicago, IL) was used for the statistical analyses. The significance level was established at P b 0.05.
Table 1 Primer sequences used for quantitative real-time PCR reactions.
CD95
CD95 ligand
Caspase 8
Caspase 3
β-Actin
Primer sequences
Annealing temperature
Base pair product
Forward primer: 5-CGT GCA CAG AAGGGG AGG AG-3 Reverse primer: 5-ACATTCATTGGCACACTTTCA GGA-3 Forward primer: 5-ACTACCACCGCC TTC CCAACC-3 Reverse primer: 5-GGC CGC CTT TCT TAT ACT TCACTC-3 Forward primer: 5-CCCCACCCTCACTTTGCT-3 Reverse primer: 5-GGAGGACCAGGCACTTA-3 Forward primer: 5-CAGAGCTGGACTGCGGTATTGA-3 Reverse primer: 5-AGCATGGCGCAAAGTGACTG-3 Forward primer: 5-GAG AGG GAA ATC GTG CGT GAC-3 Reverse primer: 5-CAT CTG CTG GAA GGT GGA CA-3
56 °C
399
59 °C
368
60 °C
303
60 °C
320
55 °C
453
Please cite this article as: Mohamed, R.H., et al., Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.02.015
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R.H. Mohamed et al. / Gene xxx (2015) xxx–xxx
3. Results 3.1. Sperm characteristics Epididymal sperm concentration, sperm motility, and abnormal sperm rates were presented at Table 2. DOX treatment caused significant (P b 0.01) decreases in sperm concentration and motility; it increased significantly the head, tail and total abnormality rates of sperm (P b 0.001) in comparison with the control group. Administration of SSCs with DOX caused a significant increase in the values of sperm concentration and motility (P b 0.001) in comparison with the DOX group. However, it caused a significant decrease in abnormal sperm rates (P b 0.001) as compared to the untreated group. 3.2. Biochemical parameters Testicular tissue lipid peroxidation levels, antioxidant enzyme activities and plasma testosterone levels are presented in Table 3. MDA and testosterone levels were significantly increased in the DOX group as compared to the control group (P ≤ 0.0001). The stem cell received group had lower levels of MDA and testosterone than the doxorubicin group (P ≤ 0.0001). A significant (P ≤ 0.0001) decrease in GSH-Px, CAT and SOD levels was observed in the DOX group in comparison with the control group. However, SSCs' administration to DOX-treated rats provided a significant (P ≤ 0.0001) increase in GSH-Px, CAT and SOD activities when compared with the values of the doxorubicin group. 3.3. Expression of CD95, CD95L, Caspase 8 and Caspase 3 genes Concerning gene expression, there was a significant increase in the CD95, CD95L, Caspase 8 and Caspase 3 genes of the doxorubicin group compared to normal one (P ≤ 0.0001), however, there was a significant decrease in these genes of the SSCs' treated group compared to the DOX group (P ≤ 0.0001) (Table 4).
in the basal cells of seminiferous tubules of the DOX-treated group (Fig. 2b) while no reaction was detected in the control group (Fig. 2a). Moreover, weak cytoplasmic reaction was observed in some cells lining seminiferous tubules of the stem cell received group (Fig. 2c). These results were confirmed by morphometrical analysis of area % of NF-κB immunoreactions (Table 5). Statistical analysis of the epithelial height of the seminiferous tubules of the DOX-treated group showed a highly significant decrease as compared with other groups. However, the stem cell received group showed a highly significant increase in their epithelial height as compared with the doxorubicin treated group (Table 5). 3.6. Electron microscope examination of seminiferous tubules (st) It was observed in Fig. 3 that the control seminiferous tubules contain Sertoli cells that rest on regular basement membrane (Fig. 3a). These cells had large euchromatic nuclei. Spermatogonia, primary spermatocytes, spermatids and mature sperms were seen. Tight junctions between sertoli cells forming blood testis barrier are intact (Fig. 3b). Cut section in mature sperms showed normal appearance in different parts of sperms in the control group (Fig. 3c). Leydig cells are of normal structure (Fig. 3d). Seminiferous tubules of the DOX-treated group appeared degenerated. Apoptotic basal cells with dense nuclei, wide separations between germinal epithelium and separation of basal cells from basement membrane together with thickened folded tubular basement membrane were observed in most of tubules (Fig. 3e, f, g). Affected sperms appeared with a distorted axoneme and mitochondrial sheath (Fig. 3h). Leydig cells appeared vacuolated and with dense bodies and lipid droplets filling cytoplasm (Fig. 3i). The epithelial cells lining seminiferous tubules of stem cell recipient groups appeared normal with all types of germinal epithelium and normal blood testis barrier. Intratubular vacuolations were observed in some of the tubules (Fig. 3j, k). Normal cut sections of mature sperms and Leydig cells were detected (Fig. 3l, m). 4. Discussion
3.4. Histological examination of the testis Examination of H&E-stained sections (Fig. 1) of specimens removed from control (Fig. 1a, b) and stem cell recipient groups (Fig. 1g, h) showed closely packed and organized seminiferous tubules (st) with little interstitium. The tubules are lined with stratified germinal epithelium and Sertoli cells. All types of cells had normal cellular attachment. Tubular lumens are filled with sperms. In contrast, accentuated damage of the seminiferous epithelium with depletion and vacuolization of the germinal epithelium, as well as appearance of darkly stained apoptotic nuclei in basal spermatogonia and Sertoli cells. Moreover, multinucleated cells were revealed in some of the tubules and also detached germ cells in the tubular lumen were frequently observed in the DOX-treated group (Fig. 1c, d, e, f). 3.5. NF-κB immunostaining Fig. 2 showed that there is a strong cytoplasmic reaction in most of the cells lining seminiferous tubules (st) and positive nuclear reaction
Drugs used for chemotherapy are well-known to produce toxic sideeffects in multiple organ systems. The most commonly affected are those organs containing self-renewing cell populations, including the reproductive system (Tomao et al., 2006). Gonadal injury, which is commonly observed following this type of treatment, has been less extensively investigated than other adverse effects of these drugs, perhaps because no life-threatening symptoms arise from damage to the gonads. Testicular toxicity and infertility after chemotherapy in young men are a well-documented phenomenon (Oktay, 2005). Several studies suggest that germ cells are targets of doxorubicin toxicity in the developing testis (Ateşşahin et al., 2006; eribaşl et al., 2012). The data presented here indicates that a dose of DOX once a week during testicular development could result in a reduced adult SSC population potentially as a result of the loss of SSCs during testis development or accelerated irreversible differentiation of SSCs into spermatogonia committed for sperm production. The differentiation of spermatozoa is maintained through the function of Leydig cells which secrete testosterone. So that a significant decrease in sperm
Table 2 Epididymal sperm concentrations, sperm motility, and abnormal sperm rates in all groups. Epididymal sperm concentration (million g−1)
Normal (n = 19) DOX (n = 10) DOX + SSCs (n = 16)
310.8 ± 11.6 92.4 ± 8.2⁎ 304.2 ± 22.6#
Sperm motility (%)
70.5 ± 1.9 34.3 ± 2.4⁎ 60 ± 2.7#
Abnormal sperm rate (%) Head
Tail
Total
2.2 ± 0.19 45.1 ± 7.07⁎ 33.2 ± 5.32#
3.5 ± 0.22 18.9 ± 1.8⁎ 15.9 ± 1.5#
5.7 ± 0.41 64 ± 8.8⁎ 49.1 ± 6.8#
⁎ Significant from control, P b 0.001. # Significant from the DOX group, P b 0.001.
Please cite this article as: Mohamed, R.H., et al., Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.02.015
R.H. Mohamed et al. / Gene xxx (2015) xxx–xxx
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Table 3 Oxidative stress biomarkers and plasma testosterone level in studied groups. MDA (nmol/g tissue) Normal (n = 19) DOX (n = 10) DOX + SSCs (n = 16)
9.2 ± 1.3
GSH-Px (IU/g protein)
CAT (IU/g tissue)
SOD (U/g tissue)
Testosterone (ng/ml)
16.3 ± 2.4
6.1 ± 0.4
17.1 ± 0.66
2.5 ± 0.1
1.7 ± 0.5⁎
7.5 ± 1.4⁎
0.1 ± 0.03⁎
15.8 ± 1.1⁎#
2.0 ± 0.32⁎#
24.2 ± 2.8⁎
6.5 ± 0.3⁎
10.2 ± 1.6#
15.7 ± 1.3#
5.3 ± 0.54⁎#
⁎ Significant from control, P ≤ 0.0001. # Significant from the DOX group, P ≤ 0.0001.
concentration, sperm motility, and testosterone levels, and a significant increase in abnormal sperm rates were observed in this group (Tables 2 & 3) together with degenerated STs and interstitial cells (Fig. 3). DOX administration resulted in excessive lipid peroxidation with concomitant decrease in glutathione peroxidase (GSH-Px), catalase, and superoxide dismutase in rat testes (Table 3) as previous studies reported (Prahalathan et al., 2004, 2005). It has been reported that doxorubicin interacts with mitochondrial NADH-dehydrogenase to facilitate the production of the doxorubicin semiquinone free radical intermediate, which in a subsequent reaction with molecular oxygen forms ROS (Mimnaugh et al., 1985). Doxorubicin induced an increase in reactive oxygen species (ROS) levels or its direct effects on Leydig cell causing its impairment leading to a decrease in testosterone production (Endo et al., 2003; Ateşşahin et al., 2006). In this study, it was observed that the reason of the DOXinduced decreased testosterone concentration was mainly due to the damages in Leydig cells (Fig. 3). Because spermatozoa plasma membranes contain large quantities of polyunsaturated fatty acids and their cytoplasm contains low concentrations of scavenging enzymes, they are particularly susceptible to the damage induced by excessive ROS (Aitken and McLaughlin, 2007). Thus, the increase of free radicals in cells can induce the lipid peroxidation by oxidative breakdown of polyunsaturated fatty acids in membranes of cells. Obviously, peroxidation of sperm lipids destroys the structure of lipid matrix in the membranes of spermatozoa, and it is associated with rapid loss of intracellular ATP leading to axonemal damage, decreased sperm viability and increased mid-piece morphological defects, and it even completely inhibits spermatogenes in extreme cases (Türk et al., 2010). It has been reported that DOX-induced direct DNA fragmentation (Suominen et al., 2003), chromosomal aberrations (Au and Hsu, 1980), and oxidative stress (Prahalathan et al., 2005; Ateşşahin et al., 2006) causing a decrease in sperm count and motility as well as an increase in dead and abnormal sperm rates. Previous studies demonstrated that anticancer drugs induce apoptosis by a mitochondrial pathway that is independent of death receptors (Engels et al., 2000). The reason for the controversial role of CD95 in drug-induced cell death is currently unclear. Therefore, we investigated CD95, CD95L, Caspase 8, and Caspase 3 expression levels in testicular tissue in order to determine the possible mechanism of DOX-induced testis injury.
Table 4 Effect of SSCs on the expression of CD95, CD95L, Caspase-8 and Caspase-3 in the studied groups.
Normal (n = 19) DOX (n = 10) DOX + SSCs (n = 16)
CD95
CD95L
Caspase-3
Caspase-8
2.1 ± 0.1
1.1 ± 0.04
1.4 ± 0.05
0.8 ± 0.03
8.7 ± 0.9⁎
3.6 ± 0.2⁎
3.7 ± 0.2⁎
2.7 ± 0.5⁎
2.8 ± 0.3#
1.9 ± 0.15⁎#
1.6 ± 0.3#
1.3 ± 0.1#
⁎ Significant from control, P ≤ 0.0001. # Significant from the DOX group, P ≤ 0.0001.
We found that overexpression of CD95, CD95L, Caspase 8, and Caspase 3 genes in testicular tissues was observed in the DOX group compared to normal one (Table 4). This data is in accordance with previous studies (Massart et al., 2004; Kalivendi et al., 2005). CD95 was isolated as a type I transmembranous glycoprotein and is known to be expressed abundantly and to mediate apoptosis in a variety of immune and non-immune tissues (Watanabe-Fukunaga et al., 1992). CD95L was discovered and identified as a type II transmembranous protein belonging to the TNF superfamily, and it very efficiently triggers apoptosis in various cell types by coupling with the cell surface receptor CD95 (Nagata and Golstein, 1995). The testis represents the major non-lymphoid source of CD95L in the body. Testis toxicity may therefore be through Sertoli cells, or “nurse cells”, which form the blood–testis barrier by virtue of tight junctions, maintain homeostasis, provide nourishment for the developing sperm cells, and protect germ cells from toxic injury. Degradation of the support capacity of Sertoli cells and increased expression of CD95L on Sertoli cells and CD95 on germ cells consequently cause robust apoptosis in germ cells (Richburg, 2000). Activation of receptor upon ligand binding leads to the formation of a death-inducing signaling complex (DISC) consisting of the adaptor protein Fas-associated death domain (FADD) and pro-Caspase-8. In this process, pro-Caspase-8 is self-cleaved, activated, and released into the cytoplasm and in turn activates the effector Caspase (Caspase 3) responsible for the proteolytic degradation of structural and signaling proteins that eventually results in cell death (Brunelle and Zhang, 2010). Another mechanism of cell death depends on activation of NF-κB, which is a major transcription factor known to regulate numerous apoptosis-related genes (Rasoulpour and Boekelheide, 2005). Generation of oxidative stress due to DOX treatment is responsible for the activation of a redox-sensitive transcription factor, NF-κB (Li and Karin, 1999). Under nonstimulating conditions, NF-κB is inhibited by specific inhibitors that masks its nuclear localization and causes its retention in the cytoplasm. Activation of NF-κB occurs when these inhibitors are phosphorylated and degraded by TNF-α or IL-1β, thus releasing NF-κB for transport to the nucleus (Senftleben and Karin, 2002). In the rat testis, the NF-κB proteins are present in Sertoli cell and spermatocyte which may play a role in the regulation of stage-specific gene expression during rat spermatogenesis (Delfino and Walker, 1998). In the present investigation, strong nuclear and cytoplasmic immunohistochemical expression of NF-κB was detected in the DOX treated group as compared to the control group (Fig. 2). Thus, it can be inferred that DOX-induced oxidative stress led to an increase in the expression of NF-κB, which in turn, resulted in an enhanced expression of various genes like Caspase-3 leading to apoptosis in the testicular cells. The current histopathological examination of testicular tissues revealed that the doxorubicin treated group showed an increase in disorganization, vacuolization in both SNTs and in the germinal epithelium. Some of the tubules were found to be atrophied and deprived of the cellular structure within, which indicated severe germ cell toxicity induced by DOX (Fig. 1). Similar impaired spermatogenesis has been
Please cite this article as: Mohamed, R.H., et al., Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.02.015
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Fig. 1. H&E stained section of testes of experimental groups. Control group (1a, b): showing regular seminiferous tubules (st), narrow intertubular space with interstitial cell nuclei (arrow head), tubules are lined by stratified germinal epithelium (g) with spermatogonia (1), spermatid (2), sperms (3) and Sertoli (4). The doxorubicin-treated rats (1c, d, e, f): showing distorted seminiferous tubules (st) with disorganized germinal epithelial layers, wide intertubular spaces (s) and vacuolated (v) seminiferous tubules are seen in (c, d). Irregular atrophied tubules with a depleted germinal epithelium and sloughing of some cells into the lumen (L) are seen in (d). Darkly stained nuclei (arrows) of the basal spermatogonia and Sertoli cells are also seen in most of the tubules. (1f), multinuclear giant cells (curved arrow) in the lumen of the tubules. Stem cell recipient group (1g, h): Normal seminiferous tubules of the stem cell recipient group. The adjacent tubules are lined with many layers of germinal epithelium with intercellular vacuolations (v). The tubular lumen is filled with sperm (sp).
reported in DOX administered rat testes (Yeh et al., 2009). Also, degenerated Sertoli cells with disrupted tight junctions suggest that DOX damages immature Sertoli cells. Disruption of Sertoli cell function by toxicants can reduce their capacity to nurse germ cells, leading to apoptosis of the latter (Richburg, 2000). In those patients whose anticancer therapy clinically predicts a complete depletion of SSCs, the outlined approaches of germ cell transplantation and testicular grafting might offer options for fertility preservation. SSCs and their ability to self-renew are critical for maintenance of male fertility by supplying differentiating cells for sperm production in mammals. A technique for transplanting SSCs was first
described by Brinster and colleagues (Brinster and Zimmermann, 1994; Brinster and Avarbock, 1994), and it has become the “gold standard” for assessing stem cell activity in rodent testes. Restoration of fertility following SSCs' transplantation in rodents is suggesting therapeutic potential for the technique in humans. Here, we are showing that SSCs' injection to DOX rat resulted in a rapid reversal of tissue damage. Restoration of normal histological structure of testis was evident with the appearance of all germ cells including mature sperms with normal structure (Fig. 1). A previous study found that the donor germ cell suspension remains widely dispersed within the recipient seminiferous tubules during the
Fig. 2. Immunohistochemical reaction of NF-kB in seminiferous tubules showing negative reaction in the control group (a), strong nuclear reaction (arrow head) and cytoplasmic reaction in Sertoli cell (curved arrow) is seen in the adriamycin treated group (b). Weak positive immune reaction is seen in few cells of some SNTs (arrow) of the stem cell received group (c).
Please cite this article as: Mohamed, R.H., et al., Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.02.015
R.H. Mohamed et al. / Gene xxx (2015) xxx–xxx Table 5 Epithelial heights in seminiferous tubules and immunohistochemical reaction of NF-κB of studied groups.
Control DOX DOX + SSCs
Epithelial heights
Area % NF-κB
83.4 ± 0.5 70.8 ± 0.29⁎ 82.1 ± 3.9#
2.97 ± 0.25 8.2 ± 0.56⁎ 1.37 ± 0.10#
⁎ Significant from control, P ≤ 0.0001. # Significant from the DOX group, P ≤ 0.0001.
first weeks following transplantation. At 4 week post transplantation, donor germ cells are divided on the basement membrane of the recipient seminiferous tubuli, forming a network. Differentiation of donor germ cells to more mature germ cell stages appears to begin after this initial colonization and spreading on the recipient seminiferous tubule basement membrane (Nagano et al., 1998).
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In the present work, SSCs' injection to DOX rat caused a diminished tissue level of CD95, CD95L, Caspase 8, Caspase 3 and immune expression of NF-κB, moreover, elevated production of antioxidant enzymes in DOX/SSCs' rat was observed. When SSCs were harvested from donor testes and transplanted into a sterilized recipient testis, morphologically and functionally normal spermatogenesis was reestablished. These results are consistent with previous findings by others (Lo et al., 2005; Becker and Jakse, 2007) that revealed a successful transplantation of SSCs and that Leydig cell progenitors conserve the fertility and antiapoptotic effect of SSCs. Our data revealed that SSCs can be a potent candidate therapeutic treatment for infertility. 5. Conclusion In the present study, we observed that spermatogonial stem cell prevented DOX-induced activation of intrinsic apoptotic signaling
Fig. 3. Electron micrograph of seminiferous tubules showing the control group (3a, b, c, d): Sertoli cell (S) resting on thin regular basement membrane (B), spermatogonia (sg), 1ry spermatocytes and spermatids (st) and normal tight junction between Sertoli cells (double arrows). (3c): Cut section of the mature sperms (arrows) appears normal. Observe the axoneme, mitochondrial sheath and fibrous sheath. (3d): Leydig cells with large irregular nucleus and mitochondria with tubular cristae (curved arrows). (3e, f, g, h, i): Seminiferous tubules of DOXtreated group, germ cells (g) are widely separated and large heterogeneous bodies (H) are seen. Spermatogonia (sg) are separated (stars) from underlying basement membrane (B). (3f): Sertoli cell nucleus (Sn) is deformed (arrow head) and its cytoplasm contains vacuoles (v). (3g): Apoptotic basal cell with dense nucleus (d), disrupted cellular junctions (star) and thickened infolded tubular basement membrane (B). (3h): Affected sperms (arrows) appear with distorted axoneme and mitochondrial sheath. (3i): Leydig cells' cytoplasm appears vacuolated (V) and contains dense bodies (d) and lipid droplets (L). Seminiferous tubules of stem cell recipient group (3j, k, l, m): appears normal with Sertoli cell (S), primary spermatocyte (1S) resting on thin regular basement membrane (B) and normal tight junction between Sertoli and other germ cells (double arrows). Normal organized cut section of sperms is seen in (3l). Leydig cell is also normal with some vacuoles (3m).
Please cite this article as: Mohamed, R.H., et al., Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.02.015
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pathway. Specifically, SSCs prevented DOX-induced increases in CD95 and CD95L levels and cleaved Caspase-8 and Caspase-3 levels in testicular tissues. In addition, spermatogonial stem cell decreased Doxinduced nuclear factor-kappa B (NF-κB) activation. Finally, it should be noted that SSCs may also offer a renewable source of cells to be used to correct many diseases of aging, to develop new cell-based therapies, and to advance germline gene therapy. Conflict of interest The authors declare no conflict of interest. Acknowledgments This study was funded with the support of academic research in Zagazig University Projects, Zagazig University Post Graduate & Research Affairs (4k1/M5). References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Aitken, R.J., McLaughlin, E.A., 2007. Molecular mechanisms of sperm capacitation: progesterone-induced secondary calcium oscillations reflect the attainment of a capacitated state. Soc. Reprod. Fertil. Suppl. 63, 273–293. Ateşşahin, A., Karahan, I., Türk, G., Gür, S., Yılmaz, S., Çeribas, A.O., 2006. Protective role of lycopene on cisplatin induced changes in sperm characteristics, testicular damage and oxidative stress in rats. Reprod. Toxicol. 21, 42–47. Au, W.W., Hsu, T.C., 1980. The genotoxic effects of adriamycin in somatic and germinal cells of the mouse. Mutat. Res. 79, 351–361. Bancroft, J., Gamble, A., 2002. Theory and Practice of Histological Techniques. 5th ed. Churchill Livingstone, New York, London, pp. 165–180. Becker, C., Jakse, G., 2007. Stem cells for regeneration of urological structures. Eur. Urol. 51, 1217–1228. Brinster, R.L., Avarbock, M.R., 1994. Germline transmission of donor haplotype following spermatogonial transplantation. Proc. Natl. Acad. Sci. 91, 11303–11307. Brinster, R.L., Zimmermann, J.W., 1994. Spermatogenesis following male germ-cell transplantation. Proc. Natl. Acad. Sci. 91, 11298–11302. Brunelle, J.K., Zhang, B., 2010. Apoptosis assays for quantifying the bioactivity of anticancer drug products. Drug Resist. Updat. 13, 172–179. Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. Methods Enzymol. 52, 302–310. Carter, J., Rowland, K., Chi, D., Brown, C., Abu-Rustum, N., Castiel, M., 2005. Gynecologic cancer treatment and the impact of cancer-related infertility. Gynecol. Oncol. 97, 90–95. eribaşl, A.O., Sakin, F., Türk, G., Sönmez, M., Ateşşahin, A., 2012. Impact of ellagic acid on adriamycin-induced testicular histopathological lesions, apoptosis, lipid peroxidation and sperm damages. Exp. Toxicol. Pathol. 64, 717–724. Çeribaşl, A.O., Türk, G., Sönmez, M., Sakin, F., Ateşşahin, A., 2010. Toxic effect of cyclophosphamide on sperm morphology, testicular histology and blood oxidant–antioxidant balance, and protective roles of lycopene and ellagic acid. Basic Clin. Pharmacol. Toxicol. 107, 730–736. Delfino, F., Walker, W.H., 1998. Stage-specific nuclear expression of NF-κB in mammalian testis. Mol. Endocrinol. 12, 1696–1707. Endo, F., Manabe, F., Takeshima, H., Akaza, H., 2003. Protecting spermatogonia from apoptosis induced by doxorubicine using the luteinizing hormone-releasing hormone analog leuprorelin. Int. J. Urol. 10, 72–77. Engels, I.H., Stepczynska, A., Stroh, C., Lauber, K., Berg, C., Schwenzer, R., Wajant, H., Ja-Ènicke, R.U., Porter, A.G., Belka, C., Gregor, M., Schulze-Ostho, K., Wesselborg, S., 2000. Caspase-8/FLICE functions as an executioner caspase in anticancer druginduced apoptosis. Oncogene 19, 4563–4573. Glauert, A.M., Lewis, P.R., 1998. Biological Specimen Preparation for Transmission Electron Microscopy. 1st ed. Princeton University Press, London. Hannun, Y.A., 1997. Apoptosis and the dilemma of cancer chemotherapy. Blood 89, 1845–1853. Herrid, M., Olejnik, J., Jackson, M., Suchowerska, N., Stockwell, S., Davey, R., Hutton, K., Hope, S., Hill, J.R., 2009. Irradiation enhances the efficiency of testicular germ cell transplantation in sheep. Biol. Reprod. 81, 898–905. Hermann, B.P., Sukhwani, M., Winkler, F., Pascarella, J.N., Peters, K.A., Sheng, Y., Valli, H., Rodriguez, M., Ezzelarab, M., Dargo, G., Peterson, K., Masterson, K., Ramsey, C., Ward, T., Lienesch, M., Volk, A., Cooper, D.K., Thomson, A.W., Kiss, J.E., Penedo, M.T., Schatten, G.P., Mitalipov, S., Orwig, K.E., 2012. Spermatogonial stem cell transplantation into Rhesus testes regenerates spermatogenesis producing functional sperm. Cell Stem Cell 11, 715–726. Howlader, N., Noone, A.M., Krapcho, M., 2011. SEER Cancer Statistic Review 1975–2008. National Cancer Institute, Bethesda, Maryland, USA. Kato, M., Makino, S., Kimura, H., Ota, T., Furuhashi, T., Nagamura, Y., 2001. Sperm motion analysis in rats treated with adriamycin and its applicability to male reproductive toxicity studies. J. Toxicol. Sci. 26, 51–59.
Kalivendi, S.V., Konorev, E.A., Cunningham, S., Vanamala, S.K., Kaji, E.H., Joseph, J., Kalyanaraman, B., 2005. Doxorubicin activates nuclear factor of activated Tlymphocytes and Fas ligand transcription: role of mitochondrial reactive oxygen species and calcium. Biochem. J. 389, 527–539. Keros, V., Hultenby, K., Borgström, B., Fridström, M., Jahnukainen, K., Hovatta, O., 2007. Methods of cryopreservation of testicular tissue with viable spermatogonia in pre-pubertal boys undergoing gonadotoxic cancer treatment. Hum. Reprod. 22, 1384–1395. Kiernan, J.A., 2000. Histological and histochemical methods. Theory and Practice, 3rd ed. Butterworth Hememann/Johanne Shurp, Oxford, Auckland, Boston/Melbourne, New Delln, pp. 267–281 (390–418). Lawrence, R.A., Burk, R.F., 1976. Glutathione peroxidase activity in selenium-deficient rat liver. Biochem. Biophys. Res. Commun. 71, 952–958. Lee, S.J., Schover, L.R., Partridge, A.H., Patrizio, P., Wallace, W.H., Hagerty, K., Beck, L.N., Brennan, L.V., Oktay, K., 2006. American Society of Clinical Oncology recommendations on fertility preservation in cancer patients. J. Clin. Oncol. 24, 2917–2931. Li, N., Karin, M., 1999. Is NF-kappa B the sensor of oxidative stress? FASEB J. 13, 1137–1143. Lo, K.C., Whirledge, S., Lamb, D.J., 2005. Stem cells: implications for urology. Curr. Urol. Rep. 6, 49–54. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 193, 265–275. Massart, C., Barbet, R., Genetet, N., Gibassier, J., 2004. Doxorubicin induces Fas-mediated apoptosis in human thyroid carcinoma cells. Thyroid 14, 263–270. Mikkola, M., Sironen, A., Kopp, C., Taponen, J., Sukura, A., Vilkki, J., Katila, T., Andersson, M., 2006. Transplantation of normal boar testicular cells resulted in complete focal spermatogenesis in a boar affected by the immotile short-tail sperm defect. Reprod. Domest. Anim. 41, 124–128. Mimnaugh, E.G., Trush, M.A., Bhatnagar, M., Gram, T.E., 1985. Enhancement of reactive oxygen-dependent mitochondrial membrane lipid peroxidation by the anticancer drug adriamycin. Biochem. Pharmacol. 34, 847–856. Nagata, S., Golstein, P., 1995. The Fas death factor. Science 267, 1449–1456. Nagano, M., Avarbock, M.R., Leonida, E.B., Brinster, C.J., Brinster, R.L., 1998. Culture of mouse spermatogonial stem cells. Tissue Cell 30, 389–397. Ogawa, T., Arechaga, J.M., Avarboc, M.R., Brinster, R.L., 1997. Transplantation of testis germinal cells into mouse seminiferous tubules. Int. J. Dev. Biol. 41, 111–122. Oktay, K., 2005. Fertility preservation: an emerging discipline in the care of young patients with cancer. Lancet Oncol. 6, 192–193. Pentikainen, V., Suomalainen, L., Erkkila, K., Martelin, E., Parvinen, M., Markku, O., Dunkel, L., 2002. Nuclear factor-κB activation in human testicular apoptosis. Am. J. Pathol. 160, 205–218. Prahalathan, C., Selvakumar, E., Varalakshmi, P., 2004. Remedial effect of DL-α-lipoic acid against adriamycin induced testicular lipid peroxidation. Mol. Cell. Biochem. 267, 209–214. Prahalathan, C., Selvakumar, E., Varalakshmi, P., 2005. Protective effect of lipoic acid on adriamycin-induced testicular toxicity. Clin. Chim. Acta 360, 160–166. Rasoulpour, R.J., Boekelheide, K., 2005. NF-κB is activated in the rat testis following exposure to mono-(2-ethylhexyl) phthalate. Biol. Reprod. 72, 479–486. Richburg, J.H., 2000. The relevance of spontaneous-and chemically-induced alterations in testicular germ cell apoptosis to toxicology. Toxicol. Lett. 15, 79–86. Ries, L.A.G., Melbert, D., Krapcho, M., Mariotto, A., Miller, B.A., Feuer, E.J., Clegg, L., Horner, M.J., Howlader, N., Eisner, M.P., Reichman, M., Edwards, B.K., 2007. SEER Cancer Statistics Review, 1975–2004. National Cancer Institute, Bethesda, Maryland, USA. Senftleben, U., Karin, M., 2002. The IKK/NF-kappa B pathway. Crit. Care Med. 30, S18–S26. Sönmez, M., Türk, G., Yüce, A., 2005. The effect of ascorbic acid supplementation on sperm quality, lipid peroxidation and testosterone levels of male Wistar rats. Theriogenology 63, 2063–2072. Steliarova-Foucher, E., Stiller, C., Kaatsch, P., Berrino, F., Coebergh, J.W., Lacour, B., Parkin, M., 2004. Geographical patterns and time trends of cancer incidence and survival among children and adolescents in Europe since the 1970s (the ACCIS project): an epidemiological study. Lancet 364, 2097–2105. Suominen, J.S., Linderborg, J., Nikula, H., Hakovirta, H., Parvinen, M., Toppari, J., 2003. The effects of mono-2-ethylhexyl phathalate, adriamycin and N-ethyl-N-nitrosourea onstage-specific apoptosis and DNA synthesis in the mouse spermatogenesis. Toxicol. Lett. 143, 163–173. Sun, Y., Oberley, L.W., Li, Y., 1988. A simple method for clinical assay of superoxide dismutase. Clin. Chem. 34, 497–500. Tomao, F., Miele, E., Spinelli, G.P., Tomao, S., 2006. Anticancer treatment and fertility effects. Literature review. J. Exp. Clin. Cancer Res. 25, 475–481. Trivedi, P.P., Tripathi, D.N., Jena, G.B., 2011. Hesperetin protects testicular toxicity of doxorubicin in rat: role of NF kappa B, p38 and caspase-3. Food Chem. Toxicol. 49, 838–847. Türk, G., Çeribaş, A.O., Sakin, F., Sönmez, M., Ateşşahin, A., 2010. Antiperoxidative and anti-apoptotic effects of lycopene and ellagic acid on cyclophosphamide-induced testicular lipid peroxidation and apoptosis. Reprod. Fertil. Dev. 22, 587–596. Watanabe-Fukunaga, R., Brannan, C.I., Itoh, N., Yonehara, S., Copeland, N.G., Jenkins, N.A., Nagata, S., 1992. The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen. J. Immunol. 148, 1274–1279. Yeh, Y.C., Liu, T.J., Wang, L.C., Lee, H.W., Ting, C.T., Lee, W., Hung, C.J., Wang, K.Y., Lai, H.C., Lai, H.C., 2009. A standardized extract of Ginkgo biloba suppresses doxorubicininduced oxidative stress and p53-mediated mitochondrial apoptosis in rat testes. Br. J. Pharmacol. 156, 48–61. Yokoi, K., Uthus, E.O., Nielsen, F.H., 2003. Nickel deficiency diminishes sperm quantity and movement in rats. Biol. Trace Elem. Res. 93, 141–153.
Please cite this article as: Mohamed, R.H., et al., Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.02.015