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Effect of short- and medium-term toxicity of doxorubicin on spermatogenesis in adult Wistar rats
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Original article
Effect of short- and medium-term toxicity of doxorubicin on spermatogenesis in adult Wistar rats ⁎
Robson Campos Silvaa, , Daniella Maria Coelho Brittoa, Wagner de Fátima Pereirab, Gustavo Eustáquio Alvin Brito-Melob, Cristiane Tolentino Machadoa, Marcelo Mattos Pedreirac a
Laboratory of Cellular Biology, Department of Basic Sciences, Federal University of Jequitinhonha and Mucuri Valleys,Alto da Jacuba, Diamantina, Minas Gerais, Brazil Immunology Laboratory, Integrated Health Research Center, Federal University of Jequitinhonha and Mucuri Valleys, Alto da Jacuba, Diamantina, Minas Gerais, Brazil c Department of Animal Sciences, Federal University of Jequitinhonha and Mucuri Valleys, Alto da Jacuba, Diamantina, Minas Gerais, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Rat Testis Spermatogenesis Stereology Doxorubicin
Doxorubicin (DXR) is a widely used chemotherapeutic anticancer agent that has potent activity against several solid and non-solid human malignant tumors, including childhood malignancies. However, DXR has serious toxic effects on tissues with rapid cell cycles, such as myeloid and lymphatic tissues, intestinal mucosa, testes and ovaries. In the present study, the short- and medium-term toxic effects of DXR on the reproductive system of male Wistar rats were evaluated using morphometric and stereological tools to quantify damage to the seminiferous epithelium. Adult male Wistar rats were treated with dose of 7.5 mg/kg of DXR and were sacrificed at seven, 14, 21 and 28 days after treatment. The testes were fixed in glutaraldehyde solution, routinely processed and embedded in plastic for evaluation under a light microscope. A significant reduction in testis weight was found as a result of massive germ cell apoptosis. Differences in comparison to the control group were found in the relative frequency of all stages of the seminiferous epithelium cycle, with significant differences for stages VIII–XI. Apoptosis significantly decreased the number of pachytene spermatocytes in the stages evaluated (I, II–III and VIII) at seven and 14 days. At 21 and 28 days after treatment, the testes exhibited the massive loss of germ cells that resulted in a missing cell layer. Moreover, reductions in the height of seminiferous tubules, tubular diameter and tubular compartment as well as an increase in the intertubular compartment were found in the period studied.
1. Introduction The chemotherapeutic agent doxorubicin (DXR) or AdriamycinTR is a widely used in the treatment of cancer. DXR is a glycoside antibiotic belonging to anthracycline family obtained from Streptomyces peucetius var. caesius [1] with potent chemotherapeutic activity against several solid and non-solid human malignant tumors, including childhood malignancies [2,3]. The major mechanisms involved in DOX-induced testicular toxicity include oxidative stress resulting from lipid peroxidation and cellular apoptosis [4]. Although the antitumor action of DXR is mediated by a large number of mechanisms, oxidative stress and the generation of toxic reactive oxygen species are the main causes of its toxicity [5]. In rats and mice, DXR is rapidly removed from the plasma after injection and deposited in tissues. The drug is mainly excreted through bile and moderately excreted through urine [6]. In some rodents, DXR accumulates in the kidneys, liver, heart, and small intestine with
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greater intensity than daunorubicin, which is another member of anthracycline family [6]. Studies have indicated that DXR has the ability to induce mutations and chromosomal aberrations in normal and malignant cells [7–9]. In a similar manner to other chemotherapeutic agents, DXR has serious toxic effects on tissues with rapid cell cycles, such as myeloid and lymphatic tissues, intestinal mucosa, testes and ovaries [5]. The prognosis of childhood cancer has improved markedly in the last 20 years. However, the long life expectancy makes young survivors especially prone to the late onset consequences of successful cancer therapy, such as therapy-induced secondary malignancies, cardiac toxicity, and infertility [10]. The impact of chemotherapy on fertility and gamete quality is of concern to patients in the reproductive age [11]. Infertility is an unfortunate side effect of some cancer therapies that impacts the quality of life of survivors who are in their reproductive or pre-reproductive years [12]. The DXR-induced deterioration of sperm motion, sperm content and
Corresponding author. E-mail address: [email protected] (R.C. Silva).
https://doi.org/10.1016/j.repbio.2018.03.002 Received 29 November 2017; Received in revised form 24 February 2018; Accepted 13 March 2018 1642-431X/ © 2018 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Silva, R.C., Reproductive Biology (2018), https://doi.org/10.1016/j.repbio.2018.03.002
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glutaraldehyde into the vessels without removing the needle. The animals were perfused for 25–30 min, immediately after which the testes were removed and weighed, then cut longitudinally by hand with a razor blade into small fragments. These fragments were immersed in 4% buffered glutaraldehyde for 12 h. Tissue samples measuring 2–3 mm in thickness were routinely processed and embedded in plastic (glycol methacrylate) for histological and stereological analyses.
sperm morphology, which were responsible for the adverse effect on male fertility [13] and the major mechanisms known to be involved in DOX-induced testicular toxicity include oxidative stress resulting from lipid peroxidation and cellular apoptosis [4]. Spermatogenesis is a highly coordinated cyclic process that begins with mitotic spermatogonial proliferation, proceeds through two meiotic divisions, and is followed by spermiogenesis, in which haploid spermatids develop into spermatozoa. Spermatogenesis involves different germ cell associations and is one of the most productive, selfrenewing systems in the body, lasting from 30 to 75 days in mammals [14]. The stages of the cycle of seminiferous epithelium are characterized according to changes in the shape of the spermatid nucleus, the occurrence of meiotic divisions, and the arrangement of spermatids within the germinal epithelium [15]. The characterization of the stages of spermatogenesis and the estimation of the length of the spermatogenic are fundamental to determining spermatogenic efficiency and performing comparative studies among species [16]. Thus, the stages and cycle duration are the essential basis for understanding normal and abnormal spermatogenesis. In rats, this cycle of the seminiferous epithelium was divided into 14 stages [17], with the subsequent publication of additional guides for staging in rats [14,18]. Previous studies involving rats have revealed that cytostatic drugs are able to induce apoptosis in the spermatogenic epithelium and the cell types most sensitive to this phenomenon are spermatogonia, zygotene spermatocyte, early pachytene spermatocytes, primary and secondary spermatocytes [19,20]. This apoptotic pathway has been associated with increased levels of caspase 9, 3, 8, 12, Fas and Bid as well as a disturbance in the Bcl-2 family protein balance [21]. The aim of the present study was to investigate the short- and medium-term effects of exposure to DXR on male reproduction in rats, emphasizing the impact of DRX on the apoptosis of germ cells, absence of the germ cell layer and the frequency of the stages of the seminiferous epithelium cycle.
2.4. Biometric data and testis stereology Sections of approximately 5 μm in thickness were obtained, placed on glass slides, and stained with hematoxylin and eosin. Counts (frequency of stages) were made with light microscopy (X 1000), based on previous studies [14,18]. Five hundred randomly selected tubules from each animal were classified in the fourteen stages at seven and 14 days after treatment with DXR. The percentage frequency of the stages was calculated by the ratio between the number of sections in each stage and the total number of sections analyzed and multiplying by 100 [23]. As pachytene spermatocytes are found in all stages of the spermatogenic cycle in rats [18], pachytene counts were performed in fifty randomly selected tubules (X 400) from each animal for comparisons between the control and experimental groups. Among the fourteen stages classified, cells were counted in the three stages with the highest frequencies (I, II–III and VIII) and consequently more reliable for statistical analysis. As it was not possible to classify the experimental group at 21 and 28 days after the injection of DXR in stages, an estimate of damaged tubules was performed by evaluating 500 randomly selected tubules (X 100). Tubular diameter and height of the seminiferous tubule epithelium were measured (X 100) using an ocular micrometer calibrated with a stage micrometer. At least thirty tubular profiles that were round or nearly round were chosen randomly and measured. The volume densities of testis tissue components were determined using a 447-intersection grid placed on the ocular of the light microscope. Fifteen randomly chosen fields (6705 points) were scored (X 400). Artifacts were rarely seen and were not included in the data. The points were classified as tubular compartment (comprising tunica propria, epithelium, and lumen) and intertubular compartment (including Leydig cells, connective tissue, blood, and lymphatic vessels).
2. Materials and methods 2.1. Animals Twenty male Wistar rats aged six to eight weeks (body weight: 250–300 g) were obtained from the animal housing facilities of the Federal University of Minas Gerais (Brazil) and kept under controlled environmental conditions with free access to food and water. The experimental protocol was approved by the local animal experimentation ethics committee (CETEA-UFMG: 99/2009).
2.5. Statistical analysis All data were submitted to normality and homoscedasticity tests. Depending on the results of the normality test, either analysis of variance ANOVA and subsequent regression or one-way ANOVA and Tukey’s test were performed using the Sigma Stat 3.5 program (Systat Software, Inc.). A p-value < 0.05 was considered indicative of statistical significance.
2.2. Experimental design The animals were divided into two groups: an experimental group (n = 16) that received a single dose (7.5 mg/kg) of doxorubicin (Eurofarma, Brazil) in the tail vein of non-anesthetized animals and a control group (n = 4) that received phosphate-buffered saline (PBS: 0.15 mol/L sodium chloride and 0.01 mol/L phosphate buffer, pH 7.4) under the same conditions. Animals were euthanized with pentobarbital at seven, 14, 21, and 28 days after treatment with DXR (experimental group) and at 28 days after PBS injection (control group).
3. Results 3.1. Biometric data Fig. 1 shows the results referring to body weight, testicular weight, and the gonadosomatic index (GSI) (testis mass divided by body weight) in the control and DRX-treated groups. No significant differences in body weight were found between the groups. However, significantly lower testis weight and GSI were found in the DXR-treated rats.
2.3. Preparation of tissue for microscopy The animals were euthanized with pentobarbital during the perfusion procedure used for fixation of the testis (n = 20). The animals were subsequently perfusion-fixed through whole body perfusion [22]. Briefly, after the intraperitoneal injection of heparin, the rats were anesthetized and the abdomen and thoracic cavity were opened to expose the heart. A needle was inserted into the left ventricle and 0.9% saline solution was used to clear the blood vessels. After clearance, a two-way valve apparatus was used to introduce 4% buffered
3.2. Testis stereology 3.2.1. Frequency of the stages of the seminiferous epithelium cycle The frequencies of all stages of the seminiferous epithelium cycle were altered in DXR-treated rats at seven and 14 days compared to the control group (Fig. 2). Significant differences between groups were found for stages VIII–XI. A significant increase in stage VIII was found 2
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Fig. 1. Body weight (g), testes weight (g) and gonadosomatic index (%) in control (day 0) and doxorubicin-treated mature Wistar rats.
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Fig. 2. Relative frequency of stages of seminiferous epithelium cycle in control and doxorubicin-treated (7 and 14 days) mature Wistar rats.
15.3% were composed of spermatogonia and elongating spermatids (Fig. 4A, C and E; Table 2). At 28 days, spermatogonia were rarer in the tubules, with 20.5% of the tubules composed of spermatogonia and round spermatids, 8.2% of the tubules exhibiting spermatogonia, round and elongating spermatids, 65.2% of the tubules composed of spermatogonia and elongating spermatids, and 6.1% of the tubules formed by Sertoli cells only (Fig. 4B, D and F; Table 2).
and significant reductions in stages IX–XI were found in DXR-treated rats compared to the control group. The DXR-treated group could not to be classified at 21 and 28 days due to the large amount of germ cell depletion. 3.2.2. Pachytene spermatocyte counts in stages I, II–III and VIII The germ cell associations normally observed in the control rats were also found in DXR-treated rats at seven and 14 days. In stage I, Sertoli cells, type A spermatogonia, pachytene cells, round spermatids in step 1 and elongated spermatids in step 15 were found. In stage II–III, Sertoli cells, type A spermatogonia, intermediate type spermatogonia, pachytene cells, round spermatids in step 2–3, and elongated spermatids in step 16 were found. In stage VIII, Sertoli cells, type A spermatogonia, preleptotene spermatocytes, pachytene cells, round spermatids in step 8 and several vacuolated residual bodies were found (Fig. 3). Pachytene spermatocytes in stages I, II–III and VIII were counted for each animal. A significantly lower number of pachytene spermatocytes in stage I was found in the DXR-treated rats at seven and 14 days compared to the control group. Moreover, a significantly lower number of pachytene spermatocytes were found in DXR-treated rats at 14 days compared to the results at seven days. No significant difference in the number of pachytene spermatocytes in stage II–III was found between the control and DXR-treated rats at seven days, but a significant reduction was found in the DXR-treated rats at 14 days. The number of pachytene spermatocytes in stage VIII was significantly lower in the DXR-treated rats at seven and 14 days compared to the control group, whereas no significant difference was found in the DXR-treated rats between the seven-day and 14-day evaluations (Table 1).
3.2.4. Tubular diameter, epithelium height and testicular parenchyma volume DXR-treated rats exhibited a significant decrease in tubular diameter and epithelium height at seven, 14, 21 and 28 days compared to the control group. Significant reductions in tubular diameter and epithelium height were also found in the experimental group (Fig. 5). Tubular compartment volume density in DXR-treated rats was significantly increased at seven days, but significantly decreased at 14, 21 and 28 days compared to the control group (Fig. 6). On the other hand, significant decreases in the intertubular compartment were found in the DXR-treated rats at seven days and significant increases among the evaluations performed in the rats at 14, 21 and 28 days when compared to the control group. The tubular and intertubular compartment also exhibited significant differences in the experimental group (at seven, 14, 21 and 28 days). 4. Discussion Chemotherapy or radiotherapy used in cancer treatment can result in temporary, long-term, or permanent gonadal toxicity in human males. The effects of cytotoxic agents are very similar in experimental laboratory animals, which provide models for elucidating the mechanisms involved [24], but rats and humans appear to be among the most sensitive [25]. The chemotherapeutic drug DXR is largely used in child cancer treatments and has shown to cause the death of healthy cells, especially those that undergo rapid proliferation, such as testicular cells, which are responsible for the formation of thousands of spermatozoa per day. Several studies have shown that DXR treatment in the pre-pubertal phase causes serious damage to the testes of adult rats [26–28]. The degree to which testicular function is affected is
3.2.3. Histological evaluation of seminiferous tubules in DXR-treated rats at 21 and 28 days The testes of DXR-treated rats at 21 and 28 days exhibited germ cell depletion and other abnormalities in the tubules, such as intraepithelial vacuoles, which were more frequent at 28 days (Fig. 4). At 21 days, 13.7% of the tubules exhibited spermatogonia, pachytene spermatocyte and elongating spermatids; 4.7% of the tubules were composed of spermatogonia and round spermatids; 66.3% of the tubules exhibited spermatogonia, round spermatids, and elongating spermatids; and 4
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Fig. 3. Testicular cross-sections of the seminiferous tubules in control (A1–A3) and doxorubicin-treated mature Wistar rats at 7 days (B1–B3) and 14 days (C1–C3) in stages I, II–III and VIII. S: Sertoli cell; A: type A spermatogonia; P: Pachytene; I: Intermediate type spermatogonia; PI: Preleptotene spermatocyte; RS: Round Spermatids; ES: Elongating Spermatids; Rb: Vacuolated residual bodies; 1 = stages I; 2–3 = stages II–III; 8 = stages VIII. Bars = 5 μm.
cycle is a strictly controlled process that is characterized by specific germ cell associations. In the present study, a common stage-related analysis was performed using criteria based on the changing morphology of the acrosomic system in young spermatids and their nuclear morphology. This cycle of the rat seminiferous epithelium was divided into 14 stages [14,17]. During the cycle, Sertoli cells change their morphology and function based on the requirements of the spermatogenic process [14]. The duration of the cycle of the seminiferous epithelium appears to be a constant for a given species and cannot be altered by natural phenomena or experimental manipulations [14,36]. However, the pattern of cell associations can be altered by experimental procedures, such as radiation or chemotherapeutic drugs, which can promote germ cell death. This classification of stages has served as the foundation for numerous studies on spermatogenesis and provides the standard by which testicular histopathology is compared [18]. In the present study, differences in the relative frequency of all stages of the seminiferous epithelium cycle were found in the DRXtreated mature Wistar rats compared to the control group. However, these differences were only significant in stages VIII–XI, with an increase in stage VIII and reductions in stages IX–XI. Changes in the distribution of spermatogenic stages have been reported in rats treated with 2,5-hexanedione [37], doxorubicin [28,38], amethopterin, cyclophosphamide, cisplatinum and 5-fluorouracil [38]. Treatment with DXR could cause transient damage in Sertoli cell function [28,38], which is likely related to alterations in the relative frequency of the stages. As Sertoli cells are responsible for the synchronization of the seminiferous epithelium cycle, alterations in these cells can cause problems with regard to the progression of the stages during the cycle. The alterations in the frequency of the stages of the seminiferous epithelium cycle after doxorubicin treatment in the present investigation were likely due to direct damage to Sertoli cells. The significant
Table 1 Pachytene spermatocytes counts in control and doxorubicin-treated (7 and 14 days) mature Wistar rats. Days control (0) 7 14 a,b
Stage I a
46.8 44.5b 7.3c
Stage II–III a
55.7 48.4a 27.6b
Stage VIII 69.8a 48.2b 53.2b
Different superscripts letters for indicate significant differences (P < 0.05).
dependent on the therapeutic agent, dose (single or fractioned), and patient age. In the present study, a single dose (7.5 mg/kg) of DXR did not alter the body weight of the rats during the experimental period, which is similar to results described in previous studies [3,26]. However, when evaluated with regard to long-term toxicity caused by DRX, the body weight of rats and mice generally decreases significantly [11,21,27,29–33], possibly due to the effects of DRX on other self-renewing systems, such as the gastrointestinal system, in which lesions are an important factor in the reduction in body weight [11]. When doses greater than 10 mg/kg are administered, animals usually die [32]. Significant reductions occurred in testes weight and the GSI in the DXR-treated rats compared to the control group, which is in agreement with previous reports [21,27,29–31,34]. The decrease in testes weight and GSI in the DRX groups was likely due to the death of germ cells during the experimental period. Indeed, previous studies report that massive germ cell loss is followed by a sharp, significant decline in testicular morphometric variables [35]. Quantitative studies on spermatogenesis are based on definitions of the seminiferous epithelial cycle stages. The seminiferous epithelium 5
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Fig. 4. Testicular cross-sections of the seminiferous tubules of doxorubicin-treated mature Wistar rats at 21 days (A, C and E) and 28 days (B, D and F). L: Tubular lumen; S: Sertoli cell; RS: Round spermatids; ES: Elongate spermatids; P: Pachytene; Rb: Vacuolated residual bodies, * Intraepithelial vacuoles. Bars = 5 μm.
morphogenic processes that are targeted by chemotherapeutic agents. It is well established that cycling cells are far more sensitive to the effects of DXR than non-cycling cells and the degree of sensitivity in germ cells is dose dependent. Studies report the death of A1-A4, intermediate and type B spermatogonia, preleptotene, zygotene, early pachytene, midpachytene and meiotic cells in rodents due to DXR [19,38,40,41]. As pachytene spermatocytes were found in all stages of the seminiferous epithelium cycle in rats in the present investigation and previous studies [18], these germ cells were used to estimate the amount of apoptosis in younger germ cells. For such, the cells were counted in the three stages with the highest frequencies (stages I, II–III, and VIII). At seven days after DRX treatment, the mature rats exhibited a significant reduction in number of pachytene spermatocytes in stage I compared to the control group and the reduction was more evident at 14 days. This can be better understood using the backcalculation technique [14] to show the window of the effect of DRX during the development of germ cells. As the duration of the cycle of the seminiferous epithelium in Wistar rats is 12.8 days [42] and the relative frequency of each particular stage is known the most affected cells were mitotic germ cells in stages VIII–XI and IV–VI. In stages II–III, a reduction was found in the number of pachytene cells between control and DRX-treated rats at seven days and the reduction in the DRX-treated rats compared to the control group was significant at 14 days. Using backcalculation [14],
Table 2 Germ cells layers in doxorubicin-treated mature Wistar rats at 21 and 28 days. Germ cells layers (%)
21 days
28 days
Spermatogonia + Pachytene + Elongating Spermatids Spermatogonia + Round Spermatids Spermatogonia + Round + Elongating Spermatids Spermatogonia + Elongating Spermatids Sertoli Cell Only
13.7 4.7 66.3 15.3 0
0 20.5 8.2 65.2 6.1
increase in stage VIII could be a result of the failure of sperm release, as observed in rats treated with DRX [28,38], and the significant decrease in stages IX–XI could be also due to the delayed Sertoli cell release of sperm in stage VIII, as observed previously in adult rats [38]. DRX binds to and intercalates DNA strands, causing the block of the cell cycle in the G2 phase, inhibiting the activity of some nuclear proteins, such as DNA and RNA-polymerase and DNA-topoisomerase II, generating free radicals, increasing oxidative stress and the generation of toxic reactive oxygen species in cells, inducing apoptosis, and altering the activity the DNA methyl-transferase 1 [4–11,39]. In testes, DXR causes direct damage to Sertoli cells and induces apoptosis in germ cells in prepubertal and adult rats [28,38]. Germ cells are especially vulnerable, since they undergo mitotic, meiotic, synthetic, and
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sections exhibited massive germ cell loss, disorganized seminiferous epithelium, missing germ cell layers and, in some tubules, intraepithelial vacuolization of different sizes. The absence of germ cell layers was due the spermiation process of round spermatocytes in steps 1–19 that had apparently progressed normally and were released in the lumen. As stated above, the germ cells located beneath these layers (mitotic cells in stages IX–VI and meiotic cells in stages VIII–IX) were affected by DRX [40]. Using the map and backcalculation [14,38], one can see that the germ cells affected by the DRX were those that are present in the seminiferous epithelium (round and elongating spermatids), which constitute the majority of tubules (66.3%). At 28 days after DRX treatment, the appearance of the tubular sections was similar to that at 21 days, but the amount of intraepithelial vacuolization had increased. At this time, the wall of most seminiferous tubules was composed of spermatogonia and elongating spermatids (65.2%), the latter of which were likely the round spermatids observed at 21 days, whereas elongating spermatids found at 21 days were released by the spermiation process at 28 days. Another morphological alteration observed at 28 days was the presence of only Sertoli cells in 6.1% of the seminiferous tubules. As 13.7% of the seminiferous tubules were formed by spermatogonia, pachytene and elongating spermatids at 21 days and this association was not found at 28 days, the pachytene spermatocytes were formed by some spermatogonia that had survived and became round or elongating spermatids at 28 days. DRX causes harm to the gonads, leading to a reduction in testicular weight due to massive germ cell loss. This damage usually promotes a decline in testicular morphometric parameters, such as the diameter of the seminiferous tubules and seminiferous epithelium height [35]. The present findings show a significant decrease in tubular diameter and epithelium height in all DRX-treated rats when compared to the control group, which is similar to findings described in previous studies [3,26,35]. The reduction in the height of the seminiferous tubules due to DRX treatment is accompanied by a proportional increase in the intertubular compartment, which was indeed found at 14, 21 and 28 days after the DRX treatment, when testicular parenchyma volume was evaluated. This increase in the intertubular compartment may also be due to the dysfunction promoted by DRX in the endothelial cells, leading to the formation of lymphatic and/or interstitial edema [26] as a consequence of the movement of water and proteins from the vascular system into tissues due to an oxidative stress imbalance [43]. Studies with a longer experimental period should be conducted to determine whether this dose of DRX promotes the apoptosis of all germ cells and whether DRX causes irreversible damage to Sertoli cells. At
Fig. 5. Tubular diameter and epithelium height (μm) in control and doxorubicin-treated mature Wistar rats.
the cells affected by DRX were meiotic germ cells in stages VIII–IX. These data observed in stages I and II–III are in agreement with findings described in a previous study regarding two peaks of DNA synthesis (stage IV–VI and stage VIII–IX) [40]. In stage VIII, the number of pachytene spermatocytes in the DRX-treated rats was significantly lower at seven and 14 days compared to the control group. In this case, spermatogonia A and A4 in stage I were the most harmed, which is similar to findings described previously [38,41]. At 21 and 28 days after treatment with DRX, it was not possible to categorize the seminiferous epithelium cycle in stages. Different degrees of seminiferous tubule damage were noted depending on the time at which the rats were sacrificed. At 21 days after treatment, the tubular
Fig. 6. Testis parenchyma volume density counts in control and doxorubicin-treated mature Wistar rats.
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28 days, which was the last evaluation in the present study, 6.1% of the seminiferous tubules were formed only by Sertoli cells. When prepubescent Wistar rats were treated with 5 mg/kg, severe damage was caused to adult spermatogenesis, suggesting the definitive loss of fertility [27,28]. If undifferentiated spermatogonia survive the cytotoxic insults and the Sertoli cells are functioning, spermatogenesis recovery could occur naturally from stem cells. Thus, the eventual recovery of sperm production after exposure to cytotoxic insults depends largely on the survival of spermatogonial stem cells as well as their functional ability to proliferate and differentiate, providing the adequate number of type A differentiating spermatogonia for the further steps in the process of spermatogenesis [44]. Based on the studies of murine spermatogenesis, the most likely scenario for spermatogonial proliferation after damage in non-primate mammals seems to involve a class of relatively rare spermatogonial stem cells (SSCs), which can be termed ‘ultimate SSCs’, an important feature of which is the greater resistance to irradiation and cytotoxic agents in comparison to transitory SSCs [45]. In conclusion, to the best of our knowledge the present data show for the first time that a dose of 7.5 mg/kg of DRX promoted a significant reduction in testis weight in rats as a result of the massive apoptosis of germ cells. Alterations were found in the relative frequency of all stages of the seminiferous epithelium cycle, with significant differences in stages VIII–XI. The number of pachytene spermatocytes was significantly reduced in DRX-treated rats in stages I, II–III and VIII at seven and 14 days. Rats evaluated at 21 and 28 days after treatment showed the massive loss of germ cells associated with the absence of germ cell layers. This large amount of apoptosis induced a reduction in the height and diameter of seminiferous tubules, accompanied by an increase in the intertubular compartment.
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Biol Reprod 2008;79:1092–101. [34] Yeh Y-C, Liu T-J, Wang L-C, Lee H-W, Ting C-T, Lee W-L, et al. A standardized extract of Ginkgo biloba suppresses doxorubicin-induced oxidative stress and p53mediated mitochondrial apoptosis in rat testes. Br J Pharmol 2009;156:48–61. [35] França LR, Russel LD. The testis of domestic animals. Male reproduction: a multidisciplinary overview. Madrid: Churchill Communications Martínez-García F, Regadera J; 1998. p. 198–219. [36] Clermont Y, Harvey SC. Duration of the cycle of the seminiferous epithelium of normal, hypophysectomized and hypophysectomized-hormone treated albino rats. Endocrinology 1965;76:80–9. [37] Chapin RE, Morgan KT, Bus JS. The morphogenesis of testicular degeneration induced in rats by orally administered 2,5-Hexanedione. Exp Mol Pathol 1983;38:149–69. [38] Russell LD, Russell JA. Short-term morphological response of the rat testis to administration of five chemotherapeutic agents. Am J Anat 1991;192:142–68. [39] Yokochi T, Robertson KD. Doxorubicin inhibits DNMT1, resulting in conditional apoptosis. Mol Pharmacol 2004;66(6):1415–20. [40] Parvinen L-M, Parvinen M. Biochemical studies of the rat seminiferous epithelial wave: DNA and RNA syntheses and effects of adriamycin. Ann Biol Biochem Biophys 1978;18:585–94. [41] Lu CC, Meistrich ML. Cytotoxic effects of chemotherapeutic drugs on mouse testis cells. Cancer Res 1979;39(9):3575–82. [42] Hilscher B, Hilscher W, Maurer W. Autoradiographische untersuchungen über den modus der proliferation und regeneration des samenepithels der Wistarratte. Z Zellforsch 1969;94:593–604. [43] Wolf MB, Baynes JW. The anti-cancer drug, doxorubicin, causes oxidant stress-induced endothelial dysfunction. Biochim Biophys Acta 2006;1760(2):267–71. [44] Albuquerque AV, Almeida FR, Weng CC, Shetty G, Meistrich ML, Chiarini-Garcia H. Spermatogonial behavior in rats during radiation-induced arrest and recovery after hormone suppression. Reproduction 2013;146:363–76. [45] de Rooij DG. The nature and dynamics of spermatogonial stem cells. Development 2017;144:3022–30.
Conflicts of interest The authors have no conflicts of interest to declare. Acknowledgments The authors are grateful to the Minas Gerais State Foundation (FAPEMIG), the Brazilian Foundation (CAPES) and Brazilian National Council for Research (CNPq) for funding this study. References [1] Chabner BA. Antineoplasic agents. In: Gilman AG, Goodman LS, editors. The pharmacological basis of therapeutics. New York: McGraw Hil; 1996. p. 932–3. [2] Mohamed RH, Karam RA, Hagrass HA, Amer MG, El-Haleem MRA. Anti-apoptotic effect of spermatogonial stem cells on doxorubicin-induced testicular toxicity in rats. Gene 2015;561:107–14. [3] Cabral REL, Okada FK, Stumpp T, Vendramini V, Miraglia SM. Carnitine partially protects the rat testis against the late damage produced by doxorubicin administered during pre-puberty. Andrology 2014;2:931–42. [4] Trivedi PP, Tripathi DN, Jena GB. Hesperetin protects testicular toxicity of doxorubicin in rat: role of NFjB, p38 and caspase-3. Food Chem Toxicol 2011;49:838–47. [5] Kiyomiya K, Matsuo S, Kurebe M. Differences in intracellular sites of action of adriamycin in neoplastic and normal differentiated cells. Cancer Chemother Pharmacol 2001;47:51–7. [6] Yesair DW, Schwartzbach E, Shuck D, Denine EP, Asbell MA. Comparative pharmacokinetics of Daunomycin and adriamycin in several animal species. Cancer Res 1972;32:1177–83. [7] Singal PK, Li T, Kumar D, Danelisen I, Iliskovic N. Adriamycin-induced heart failure: mechanism and modulation. Mol Cell Biochem 2000;207(1–2):77–86. [8] Habas K, Anderson D, Brinkworth MH. Germ cell responses to doxorubicin exposure in vitro. Toxicol Lett 2017;265:70–6. [9] Quiles J, Huertas J, Battino M, Mataix J, Ramirez-Tortosa M. Antioxidant nutrients and adriamycin toxicity. Toxicology 2002;180:79–95. [10] Jahnukainen K, Jahnukainen T, Salmi TT, Svechnikov K, Eksborg S, Söder O. Amifostine protects against early but not late toxic effects of doxorubicin in infant rats. Cancer Res 2001;61:6423–7. [11] Meistrich ML. Male gonadal toxicity. Pediatr Blood Cancer 2009;53:261–6. [12] Valli H, Phillips BT, Shetty G, Byrne JA, Clark AT, Meistrich ML, et al. Germline stem cells: toward the regeneration of spermatogenesis. Fertil Steril 2014;101(1):3–13.
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Reproductive Biology journal homepage: www.elsevier.com/locate/repbio
Original article
Effect of short- and medium-term toxicity of doxorubicin on spermatogenesis in adult Wistar rats ⁎
Robson Campos Silvaa, , Daniella Maria Coelho Brittoa, Wagner de Fátima Pereirab, Gustavo Eustáquio Alvin Brito-Melob, Cristiane Tolentino Machadoa, Marcelo Mattos Pedreirac a
Laboratory of Cellular Biology, Department of Basic Sciences, Federal University of Jequitinhonha and Mucuri Valleys,Alto da Jacuba, Diamantina, Minas Gerais, Brazil Immunology Laboratory, Integrated Health Research Center, Federal University of Jequitinhonha and Mucuri Valleys, Alto da Jacuba, Diamantina, Minas Gerais, Brazil c Department of Animal Sciences, Federal University of Jequitinhonha and Mucuri Valleys, Alto da Jacuba, Diamantina, Minas Gerais, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Rat Testis Spermatogenesis Stereology Doxorubicin
Doxorubicin (DXR) is a widely used chemotherapeutic anticancer agent that has potent activity against several solid and non-solid human malignant tumors, including childhood malignancies. However, DXR has serious toxic effects on tissues with rapid cell cycles, such as myeloid and lymphatic tissues, intestinal mucosa, testes and ovaries. In the present study, the short- and medium-term toxic effects of DXR on the reproductive system of male Wistar rats were evaluated using morphometric and stereological tools to quantify damage to the seminiferous epithelium. Adult male Wistar rats were treated with dose of 7.5 mg/kg of DXR and were sacrificed at seven, 14, 21 and 28 days after treatment. The testes were fixed in glutaraldehyde solution, routinely processed and embedded in plastic for evaluation under a light microscope. A significant reduction in testis weight was found as a result of massive germ cell apoptosis. Differences in comparison to the control group were found in the relative frequency of all stages of the seminiferous epithelium cycle, with significant differences for stages VIII–XI. Apoptosis significantly decreased the number of pachytene spermatocytes in the stages evaluated (I, II–III and VIII) at seven and 14 days. At 21 and 28 days after treatment, the testes exhibited the massive loss of germ cells that resulted in a missing cell layer. Moreover, reductions in the height of seminiferous tubules, tubular diameter and tubular compartment as well as an increase in the intertubular compartment were found in the period studied.
1. Introduction The chemotherapeutic agent doxorubicin (DXR) or AdriamycinTR is a widely used in the treatment of cancer. DXR is a glycoside antibiotic belonging to anthracycline family obtained from Streptomyces peucetius var. caesius [1] with potent chemotherapeutic activity against several solid and non-solid human malignant tumors, including childhood malignancies [2,3]. The major mechanisms involved in DOX-induced testicular toxicity include oxidative stress resulting from lipid peroxidation and cellular apoptosis [4]. Although the antitumor action of DXR is mediated by a large number of mechanisms, oxidative stress and the generation of toxic reactive oxygen species are the main causes of its toxicity [5]. In rats and mice, DXR is rapidly removed from the plasma after injection and deposited in tissues. The drug is mainly excreted through bile and moderately excreted through urine [6]. In some rodents, DXR accumulates in the kidneys, liver, heart, and small intestine with
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greater intensity than daunorubicin, which is another member of anthracycline family [6]. Studies have indicated that DXR has the ability to induce mutations and chromosomal aberrations in normal and malignant cells [7–9]. In a similar manner to other chemotherapeutic agents, DXR has serious toxic effects on tissues with rapid cell cycles, such as myeloid and lymphatic tissues, intestinal mucosa, testes and ovaries [5]. The prognosis of childhood cancer has improved markedly in the last 20 years. However, the long life expectancy makes young survivors especially prone to the late onset consequences of successful cancer therapy, such as therapy-induced secondary malignancies, cardiac toxicity, and infertility [10]. The impact of chemotherapy on fertility and gamete quality is of concern to patients in the reproductive age [11]. Infertility is an unfortunate side effect of some cancer therapies that impacts the quality of life of survivors who are in their reproductive or pre-reproductive years [12]. The DXR-induced deterioration of sperm motion, sperm content and
Corresponding author. E-mail address: [email protected] (R.C. Silva).
https://doi.org/10.1016/j.repbio.2018.03.002 Received 29 November 2017; Received in revised form 24 February 2018; Accepted 13 March 2018 1642-431X/ © 2018 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Silva, R.C., Reproductive Biology (2018), https://doi.org/10.1016/j.repbio.2018.03.002
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glutaraldehyde into the vessels without removing the needle. The animals were perfused for 25–30 min, immediately after which the testes were removed and weighed, then cut longitudinally by hand with a razor blade into small fragments. These fragments were immersed in 4% buffered glutaraldehyde for 12 h. Tissue samples measuring 2–3 mm in thickness were routinely processed and embedded in plastic (glycol methacrylate) for histological and stereological analyses.
sperm morphology, which were responsible for the adverse effect on male fertility [13] and the major mechanisms known to be involved in DOX-induced testicular toxicity include oxidative stress resulting from lipid peroxidation and cellular apoptosis [4]. Spermatogenesis is a highly coordinated cyclic process that begins with mitotic spermatogonial proliferation, proceeds through two meiotic divisions, and is followed by spermiogenesis, in which haploid spermatids develop into spermatozoa. Spermatogenesis involves different germ cell associations and is one of the most productive, selfrenewing systems in the body, lasting from 30 to 75 days in mammals [14]. The stages of the cycle of seminiferous epithelium are characterized according to changes in the shape of the spermatid nucleus, the occurrence of meiotic divisions, and the arrangement of spermatids within the germinal epithelium [15]. The characterization of the stages of spermatogenesis and the estimation of the length of the spermatogenic are fundamental to determining spermatogenic efficiency and performing comparative studies among species [16]. Thus, the stages and cycle duration are the essential basis for understanding normal and abnormal spermatogenesis. In rats, this cycle of the seminiferous epithelium was divided into 14 stages [17], with the subsequent publication of additional guides for staging in rats [14,18]. Previous studies involving rats have revealed that cytostatic drugs are able to induce apoptosis in the spermatogenic epithelium and the cell types most sensitive to this phenomenon are spermatogonia, zygotene spermatocyte, early pachytene spermatocytes, primary and secondary spermatocytes [19,20]. This apoptotic pathway has been associated with increased levels of caspase 9, 3, 8, 12, Fas and Bid as well as a disturbance in the Bcl-2 family protein balance [21]. The aim of the present study was to investigate the short- and medium-term effects of exposure to DXR on male reproduction in rats, emphasizing the impact of DRX on the apoptosis of germ cells, absence of the germ cell layer and the frequency of the stages of the seminiferous epithelium cycle.
2.4. Biometric data and testis stereology Sections of approximately 5 μm in thickness were obtained, placed on glass slides, and stained with hematoxylin and eosin. Counts (frequency of stages) were made with light microscopy (X 1000), based on previous studies [14,18]. Five hundred randomly selected tubules from each animal were classified in the fourteen stages at seven and 14 days after treatment with DXR. The percentage frequency of the stages was calculated by the ratio between the number of sections in each stage and the total number of sections analyzed and multiplying by 100 [23]. As pachytene spermatocytes are found in all stages of the spermatogenic cycle in rats [18], pachytene counts were performed in fifty randomly selected tubules (X 400) from each animal for comparisons between the control and experimental groups. Among the fourteen stages classified, cells were counted in the three stages with the highest frequencies (I, II–III and VIII) and consequently more reliable for statistical analysis. As it was not possible to classify the experimental group at 21 and 28 days after the injection of DXR in stages, an estimate of damaged tubules was performed by evaluating 500 randomly selected tubules (X 100). Tubular diameter and height of the seminiferous tubule epithelium were measured (X 100) using an ocular micrometer calibrated with a stage micrometer. At least thirty tubular profiles that were round or nearly round were chosen randomly and measured. The volume densities of testis tissue components were determined using a 447-intersection grid placed on the ocular of the light microscope. Fifteen randomly chosen fields (6705 points) were scored (X 400). Artifacts were rarely seen and were not included in the data. The points were classified as tubular compartment (comprising tunica propria, epithelium, and lumen) and intertubular compartment (including Leydig cells, connective tissue, blood, and lymphatic vessels).
2. Materials and methods 2.1. Animals Twenty male Wistar rats aged six to eight weeks (body weight: 250–300 g) were obtained from the animal housing facilities of the Federal University of Minas Gerais (Brazil) and kept under controlled environmental conditions with free access to food and water. The experimental protocol was approved by the local animal experimentation ethics committee (CETEA-UFMG: 99/2009).
2.5. Statistical analysis All data were submitted to normality and homoscedasticity tests. Depending on the results of the normality test, either analysis of variance ANOVA and subsequent regression or one-way ANOVA and Tukey’s test were performed using the Sigma Stat 3.5 program (Systat Software, Inc.). A p-value < 0.05 was considered indicative of statistical significance.
2.2. Experimental design The animals were divided into two groups: an experimental group (n = 16) that received a single dose (7.5 mg/kg) of doxorubicin (Eurofarma, Brazil) in the tail vein of non-anesthetized animals and a control group (n = 4) that received phosphate-buffered saline (PBS: 0.15 mol/L sodium chloride and 0.01 mol/L phosphate buffer, pH 7.4) under the same conditions. Animals were euthanized with pentobarbital at seven, 14, 21, and 28 days after treatment with DXR (experimental group) and at 28 days after PBS injection (control group).
3. Results 3.1. Biometric data Fig. 1 shows the results referring to body weight, testicular weight, and the gonadosomatic index (GSI) (testis mass divided by body weight) in the control and DRX-treated groups. No significant differences in body weight were found between the groups. However, significantly lower testis weight and GSI were found in the DXR-treated rats.
2.3. Preparation of tissue for microscopy The animals were euthanized with pentobarbital during the perfusion procedure used for fixation of the testis (n = 20). The animals were subsequently perfusion-fixed through whole body perfusion [22]. Briefly, after the intraperitoneal injection of heparin, the rats were anesthetized and the abdomen and thoracic cavity were opened to expose the heart. A needle was inserted into the left ventricle and 0.9% saline solution was used to clear the blood vessels. After clearance, a two-way valve apparatus was used to introduce 4% buffered
3.2. Testis stereology 3.2.1. Frequency of the stages of the seminiferous epithelium cycle The frequencies of all stages of the seminiferous epithelium cycle were altered in DXR-treated rats at seven and 14 days compared to the control group (Fig. 2). Significant differences between groups were found for stages VIII–XI. A significant increase in stage VIII was found 2
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Fig. 1. Body weight (g), testes weight (g) and gonadosomatic index (%) in control (day 0) and doxorubicin-treated mature Wistar rats.
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Fig. 2. Relative frequency of stages of seminiferous epithelium cycle in control and doxorubicin-treated (7 and 14 days) mature Wistar rats.
15.3% were composed of spermatogonia and elongating spermatids (Fig. 4A, C and E; Table 2). At 28 days, spermatogonia were rarer in the tubules, with 20.5% of the tubules composed of spermatogonia and round spermatids, 8.2% of the tubules exhibiting spermatogonia, round and elongating spermatids, 65.2% of the tubules composed of spermatogonia and elongating spermatids, and 6.1% of the tubules formed by Sertoli cells only (Fig. 4B, D and F; Table 2).
and significant reductions in stages IX–XI were found in DXR-treated rats compared to the control group. The DXR-treated group could not to be classified at 21 and 28 days due to the large amount of germ cell depletion. 3.2.2. Pachytene spermatocyte counts in stages I, II–III and VIII The germ cell associations normally observed in the control rats were also found in DXR-treated rats at seven and 14 days. In stage I, Sertoli cells, type A spermatogonia, pachytene cells, round spermatids in step 1 and elongated spermatids in step 15 were found. In stage II–III, Sertoli cells, type A spermatogonia, intermediate type spermatogonia, pachytene cells, round spermatids in step 2–3, and elongated spermatids in step 16 were found. In stage VIII, Sertoli cells, type A spermatogonia, preleptotene spermatocytes, pachytene cells, round spermatids in step 8 and several vacuolated residual bodies were found (Fig. 3). Pachytene spermatocytes in stages I, II–III and VIII were counted for each animal. A significantly lower number of pachytene spermatocytes in stage I was found in the DXR-treated rats at seven and 14 days compared to the control group. Moreover, a significantly lower number of pachytene spermatocytes were found in DXR-treated rats at 14 days compared to the results at seven days. No significant difference in the number of pachytene spermatocytes in stage II–III was found between the control and DXR-treated rats at seven days, but a significant reduction was found in the DXR-treated rats at 14 days. The number of pachytene spermatocytes in stage VIII was significantly lower in the DXR-treated rats at seven and 14 days compared to the control group, whereas no significant difference was found in the DXR-treated rats between the seven-day and 14-day evaluations (Table 1).
3.2.4. Tubular diameter, epithelium height and testicular parenchyma volume DXR-treated rats exhibited a significant decrease in tubular diameter and epithelium height at seven, 14, 21 and 28 days compared to the control group. Significant reductions in tubular diameter and epithelium height were also found in the experimental group (Fig. 5). Tubular compartment volume density in DXR-treated rats was significantly increased at seven days, but significantly decreased at 14, 21 and 28 days compared to the control group (Fig. 6). On the other hand, significant decreases in the intertubular compartment were found in the DXR-treated rats at seven days and significant increases among the evaluations performed in the rats at 14, 21 and 28 days when compared to the control group. The tubular and intertubular compartment also exhibited significant differences in the experimental group (at seven, 14, 21 and 28 days). 4. Discussion Chemotherapy or radiotherapy used in cancer treatment can result in temporary, long-term, or permanent gonadal toxicity in human males. The effects of cytotoxic agents are very similar in experimental laboratory animals, which provide models for elucidating the mechanisms involved [24], but rats and humans appear to be among the most sensitive [25]. The chemotherapeutic drug DXR is largely used in child cancer treatments and has shown to cause the death of healthy cells, especially those that undergo rapid proliferation, such as testicular cells, which are responsible for the formation of thousands of spermatozoa per day. Several studies have shown that DXR treatment in the pre-pubertal phase causes serious damage to the testes of adult rats [26–28]. The degree to which testicular function is affected is
3.2.3. Histological evaluation of seminiferous tubules in DXR-treated rats at 21 and 28 days The testes of DXR-treated rats at 21 and 28 days exhibited germ cell depletion and other abnormalities in the tubules, such as intraepithelial vacuoles, which were more frequent at 28 days (Fig. 4). At 21 days, 13.7% of the tubules exhibited spermatogonia, pachytene spermatocyte and elongating spermatids; 4.7% of the tubules were composed of spermatogonia and round spermatids; 66.3% of the tubules exhibited spermatogonia, round spermatids, and elongating spermatids; and 4
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Fig. 3. Testicular cross-sections of the seminiferous tubules in control (A1–A3) and doxorubicin-treated mature Wistar rats at 7 days (B1–B3) and 14 days (C1–C3) in stages I, II–III and VIII. S: Sertoli cell; A: type A spermatogonia; P: Pachytene; I: Intermediate type spermatogonia; PI: Preleptotene spermatocyte; RS: Round Spermatids; ES: Elongating Spermatids; Rb: Vacuolated residual bodies; 1 = stages I; 2–3 = stages II–III; 8 = stages VIII. Bars = 5 μm.
cycle is a strictly controlled process that is characterized by specific germ cell associations. In the present study, a common stage-related analysis was performed using criteria based on the changing morphology of the acrosomic system in young spermatids and their nuclear morphology. This cycle of the rat seminiferous epithelium was divided into 14 stages [14,17]. During the cycle, Sertoli cells change their morphology and function based on the requirements of the spermatogenic process [14]. The duration of the cycle of the seminiferous epithelium appears to be a constant for a given species and cannot be altered by natural phenomena or experimental manipulations [14,36]. However, the pattern of cell associations can be altered by experimental procedures, such as radiation or chemotherapeutic drugs, which can promote germ cell death. This classification of stages has served as the foundation for numerous studies on spermatogenesis and provides the standard by which testicular histopathology is compared [18]. In the present study, differences in the relative frequency of all stages of the seminiferous epithelium cycle were found in the DRXtreated mature Wistar rats compared to the control group. However, these differences were only significant in stages VIII–XI, with an increase in stage VIII and reductions in stages IX–XI. Changes in the distribution of spermatogenic stages have been reported in rats treated with 2,5-hexanedione [37], doxorubicin [28,38], amethopterin, cyclophosphamide, cisplatinum and 5-fluorouracil [38]. Treatment with DXR could cause transient damage in Sertoli cell function [28,38], which is likely related to alterations in the relative frequency of the stages. As Sertoli cells are responsible for the synchronization of the seminiferous epithelium cycle, alterations in these cells can cause problems with regard to the progression of the stages during the cycle. The alterations in the frequency of the stages of the seminiferous epithelium cycle after doxorubicin treatment in the present investigation were likely due to direct damage to Sertoli cells. The significant
Table 1 Pachytene spermatocytes counts in control and doxorubicin-treated (7 and 14 days) mature Wistar rats. Days control (0) 7 14 a,b
Stage I a
46.8 44.5b 7.3c
Stage II–III a
55.7 48.4a 27.6b
Stage VIII 69.8a 48.2b 53.2b
Different superscripts letters for indicate significant differences (P < 0.05).
dependent on the therapeutic agent, dose (single or fractioned), and patient age. In the present study, a single dose (7.5 mg/kg) of DXR did not alter the body weight of the rats during the experimental period, which is similar to results described in previous studies [3,26]. However, when evaluated with regard to long-term toxicity caused by DRX, the body weight of rats and mice generally decreases significantly [11,21,27,29–33], possibly due to the effects of DRX on other self-renewing systems, such as the gastrointestinal system, in which lesions are an important factor in the reduction in body weight [11]. When doses greater than 10 mg/kg are administered, animals usually die [32]. Significant reductions occurred in testes weight and the GSI in the DXR-treated rats compared to the control group, which is in agreement with previous reports [21,27,29–31,34]. The decrease in testes weight and GSI in the DRX groups was likely due to the death of germ cells during the experimental period. Indeed, previous studies report that massive germ cell loss is followed by a sharp, significant decline in testicular morphometric variables [35]. Quantitative studies on spermatogenesis are based on definitions of the seminiferous epithelial cycle stages. The seminiferous epithelium 5
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Fig. 4. Testicular cross-sections of the seminiferous tubules of doxorubicin-treated mature Wistar rats at 21 days (A, C and E) and 28 days (B, D and F). L: Tubular lumen; S: Sertoli cell; RS: Round spermatids; ES: Elongate spermatids; P: Pachytene; Rb: Vacuolated residual bodies, * Intraepithelial vacuoles. Bars = 5 μm.
morphogenic processes that are targeted by chemotherapeutic agents. It is well established that cycling cells are far more sensitive to the effects of DXR than non-cycling cells and the degree of sensitivity in germ cells is dose dependent. Studies report the death of A1-A4, intermediate and type B spermatogonia, preleptotene, zygotene, early pachytene, midpachytene and meiotic cells in rodents due to DXR [19,38,40,41]. As pachytene spermatocytes were found in all stages of the seminiferous epithelium cycle in rats in the present investigation and previous studies [18], these germ cells were used to estimate the amount of apoptosis in younger germ cells. For such, the cells were counted in the three stages with the highest frequencies (stages I, II–III, and VIII). At seven days after DRX treatment, the mature rats exhibited a significant reduction in number of pachytene spermatocytes in stage I compared to the control group and the reduction was more evident at 14 days. This can be better understood using the backcalculation technique [14] to show the window of the effect of DRX during the development of germ cells. As the duration of the cycle of the seminiferous epithelium in Wistar rats is 12.8 days [42] and the relative frequency of each particular stage is known the most affected cells were mitotic germ cells in stages VIII–XI and IV–VI. In stages II–III, a reduction was found in the number of pachytene cells between control and DRX-treated rats at seven days and the reduction in the DRX-treated rats compared to the control group was significant at 14 days. Using backcalculation [14],
Table 2 Germ cells layers in doxorubicin-treated mature Wistar rats at 21 and 28 days. Germ cells layers (%)
21 days
28 days
Spermatogonia + Pachytene + Elongating Spermatids Spermatogonia + Round Spermatids Spermatogonia + Round + Elongating Spermatids Spermatogonia + Elongating Spermatids Sertoli Cell Only
13.7 4.7 66.3 15.3 0
0 20.5 8.2 65.2 6.1
increase in stage VIII could be a result of the failure of sperm release, as observed in rats treated with DRX [28,38], and the significant decrease in stages IX–XI could be also due to the delayed Sertoli cell release of sperm in stage VIII, as observed previously in adult rats [38]. DRX binds to and intercalates DNA strands, causing the block of the cell cycle in the G2 phase, inhibiting the activity of some nuclear proteins, such as DNA and RNA-polymerase and DNA-topoisomerase II, generating free radicals, increasing oxidative stress and the generation of toxic reactive oxygen species in cells, inducing apoptosis, and altering the activity the DNA methyl-transferase 1 [4–11,39]. In testes, DXR causes direct damage to Sertoli cells and induces apoptosis in germ cells in prepubertal and adult rats [28,38]. Germ cells are especially vulnerable, since they undergo mitotic, meiotic, synthetic, and
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sections exhibited massive germ cell loss, disorganized seminiferous epithelium, missing germ cell layers and, in some tubules, intraepithelial vacuolization of different sizes. The absence of germ cell layers was due the spermiation process of round spermatocytes in steps 1–19 that had apparently progressed normally and were released in the lumen. As stated above, the germ cells located beneath these layers (mitotic cells in stages IX–VI and meiotic cells in stages VIII–IX) were affected by DRX [40]. Using the map and backcalculation [14,38], one can see that the germ cells affected by the DRX were those that are present in the seminiferous epithelium (round and elongating spermatids), which constitute the majority of tubules (66.3%). At 28 days after DRX treatment, the appearance of the tubular sections was similar to that at 21 days, but the amount of intraepithelial vacuolization had increased. At this time, the wall of most seminiferous tubules was composed of spermatogonia and elongating spermatids (65.2%), the latter of which were likely the round spermatids observed at 21 days, whereas elongating spermatids found at 21 days were released by the spermiation process at 28 days. Another morphological alteration observed at 28 days was the presence of only Sertoli cells in 6.1% of the seminiferous tubules. As 13.7% of the seminiferous tubules were formed by spermatogonia, pachytene and elongating spermatids at 21 days and this association was not found at 28 days, the pachytene spermatocytes were formed by some spermatogonia that had survived and became round or elongating spermatids at 28 days. DRX causes harm to the gonads, leading to a reduction in testicular weight due to massive germ cell loss. This damage usually promotes a decline in testicular morphometric parameters, such as the diameter of the seminiferous tubules and seminiferous epithelium height [35]. The present findings show a significant decrease in tubular diameter and epithelium height in all DRX-treated rats when compared to the control group, which is similar to findings described in previous studies [3,26,35]. The reduction in the height of the seminiferous tubules due to DRX treatment is accompanied by a proportional increase in the intertubular compartment, which was indeed found at 14, 21 and 28 days after the DRX treatment, when testicular parenchyma volume was evaluated. This increase in the intertubular compartment may also be due to the dysfunction promoted by DRX in the endothelial cells, leading to the formation of lymphatic and/or interstitial edema [26] as a consequence of the movement of water and proteins from the vascular system into tissues due to an oxidative stress imbalance [43]. Studies with a longer experimental period should be conducted to determine whether this dose of DRX promotes the apoptosis of all germ cells and whether DRX causes irreversible damage to Sertoli cells. At
Fig. 5. Tubular diameter and epithelium height (μm) in control and doxorubicin-treated mature Wistar rats.
the cells affected by DRX were meiotic germ cells in stages VIII–IX. These data observed in stages I and II–III are in agreement with findings described in a previous study regarding two peaks of DNA synthesis (stage IV–VI and stage VIII–IX) [40]. In stage VIII, the number of pachytene spermatocytes in the DRX-treated rats was significantly lower at seven and 14 days compared to the control group. In this case, spermatogonia A and A4 in stage I were the most harmed, which is similar to findings described previously [38,41]. At 21 and 28 days after treatment with DRX, it was not possible to categorize the seminiferous epithelium cycle in stages. Different degrees of seminiferous tubule damage were noted depending on the time at which the rats were sacrificed. At 21 days after treatment, the tubular
Fig. 6. Testis parenchyma volume density counts in control and doxorubicin-treated mature Wistar rats.
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28 days, which was the last evaluation in the present study, 6.1% of the seminiferous tubules were formed only by Sertoli cells. When prepubescent Wistar rats were treated with 5 mg/kg, severe damage was caused to adult spermatogenesis, suggesting the definitive loss of fertility [27,28]. If undifferentiated spermatogonia survive the cytotoxic insults and the Sertoli cells are functioning, spermatogenesis recovery could occur naturally from stem cells. Thus, the eventual recovery of sperm production after exposure to cytotoxic insults depends largely on the survival of spermatogonial stem cells as well as their functional ability to proliferate and differentiate, providing the adequate number of type A differentiating spermatogonia for the further steps in the process of spermatogenesis [44]. Based on the studies of murine spermatogenesis, the most likely scenario for spermatogonial proliferation after damage in non-primate mammals seems to involve a class of relatively rare spermatogonial stem cells (SSCs), which can be termed ‘ultimate SSCs’, an important feature of which is the greater resistance to irradiation and cytotoxic agents in comparison to transitory SSCs [45]. In conclusion, to the best of our knowledge the present data show for the first time that a dose of 7.5 mg/kg of DRX promoted a significant reduction in testis weight in rats as a result of the massive apoptosis of germ cells. Alterations were found in the relative frequency of all stages of the seminiferous epithelium cycle, with significant differences in stages VIII–XI. The number of pachytene spermatocytes was significantly reduced in DRX-treated rats in stages I, II–III and VIII at seven and 14 days. Rats evaluated at 21 and 28 days after treatment showed the massive loss of germ cells associated with the absence of germ cell layers. This large amount of apoptosis induced a reduction in the height and diameter of seminiferous tubules, accompanied by an increase in the intertubular compartment.
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