Reproductive Sciences

http://rsx.sagepub.com/ Histopathological and Immunohistochemical Changes in the Testes of Rabbits After Injection With the Growth Promoter Boldenone
Ehab Tousson, Mostafa El-Moghazy, Ahmed Massoud and Amani Akel Reproductive Sciences 2012 19: 253 DOI: 10.1177/1933719111418126 The online version of this article can be found at: http://rsx.sagepub.com/content/19/3/253

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Histopathological and Immunohistochemical Changes in the Testes of Rabbits After Injection With the Growth Promoter Boldenone

Reproductive Sciences 19(3) 253-259 The Author(s) 2012 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1933719111418126 http://rs.sagepub.com

Ehab Tousson, PhD1, Mostafa El-Moghazy, PhD2, Ahmed Massoud, PhD1, and Amani Akel, BSc1

Abstract Recently, boldenone (androgenic steroid) is used in improvement of the growth and food conversion in food-producing animals. In addition, it is used by bodybuilders during both off-season and precontest, where it is well known for increasing vascularity while preparing for a bodybuilding contest. The present study was designed to investigate the possible effect of growth promoter boldenone undecylenate on the structure and functions of rabbit testes. A total of 32 adult New Zealand rabbits were divided into 4 groups. The first group in the control group includes animals that were intramuscularly injected with olive oil and dissected after 3 weeks. Three experimental groups include animals that receive 1, 2, and 3 intramuscular injections of 5 mg/kg body weight boldenone, and dissected after 3, 6, and 9 weeks, respectively. Treating rabbits with boldenone increased the testosterone levels compared to the control group. Seminiferous tubules of the rabbit testis treated with boldenone showed reduced development and degeneration of the germinal epithelium, leading to debris and syncytial cell formation in the lumina of seminiferous tubules. Our immunohistochemical results indicated severe reduction in proliferating cell nuclear antigenpositive spermatogonia in boldenone-treated animals as compared to the control group. These findings explain the common phenomena among athletics and bodybuilders who suffer from infertility as they were injected with some drugs such as steroids (boldenone) to build muscles. Keywords steroids, boldenone, rabbit, testes, immunohistochemistry, PCNA

Introduction
Anabolic-androgenic steroids cause some adverse effects such as disturbance in the endocrine and immune functions, alterations of sebaceous system and skin changes in hemostatic system and urogenital tract.1-3 Anabolic-androgenic steroids are used to enhance the strength and endurance in canine, equine, and human athletes through increasing the production of muscle protein and are synthetic substances related to the primary male sex hormone, testosterone.4-8 Its role in increasing the muscle size is because of the promotion of positive nitrogen balance by stimulating protein production and reducing protein destruction. Boldenone (1,4-androstadiene-17b-ol-3one; BOL) is an androgenic steroid that improves the growth and food conversion in food-producing animals. Steroids were developed mainly for veterinary use, mostly in horse treatment. Recently, BOL is used by bodybuilders during both off-season and precontest, where it is well known for increasing vascularity while preparing for a bodybuilding contest. Boldenone is well known under the trade names Equipoise, Ganabol, Equigan, and Ultragan. In most countries worldwide, this anabolic steroid is forbidden in meat production and human use.6,9,10 It has a very long half-life and can show up on a

steroid test for up to 1.5 years. This is because of its long undecylenate ester attached to the parent steroid. Trace amounts of the drug can be easily detected for months after discontinuing its use.11,12 Both directly and indirectly, boldenone has dual effects on humans; directly as injection to build muscles and indirectly through consuming the meat of animals that where treated with BOL. Therefore, the aim of the present study was to investigate the possible effect of growth promoter boldenone undecylenate on the testicular structure and functions.

Materials and Methods

The experiment adhered to the guidelines of the ethical committee of the National Research Center, Egypt. The present
Zoology Department, Faculty of Science, Tanta University, Tanta, Egypt Animal Production Department, Faculty of Agriculture, Minoufiya University, Shebin El-Kom, Egypt
2 1

Corresponding Author: Ehab Tousson, Zoology Department, Faculty of Science, Tanta University, Tanta 00240, Egypt Email: toussonehab@yahoo.com

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254 study was conducted at a rabbit private farm in Dakhahleia governorate and Zoology Department, Faculty of Science, Tanta University, Egypt, during spring and summer 2010.

Reproductive Sciences 19(3) at room temperature until further processing. Some paraffin sections were analyzed with hematoxylin and eosin stains as a routine method, according to Bancroft and Cook15 and the rest of paraffin sections used for with proliferating cell nuclear antigen (PCNA) immunohistochemical staining method as reported by Tousson et al.16 Proliferating cell nuclear antigen immunoreactivity (PCNA-ir). Testicular distribution of PCNA receptor subunits were examined in deparaffinized sections (5 mm) using an avidinbiotin peroxidase (ABC) immunohistochemical method (Elite-ABC, Vector Laboratories, California) and anti-PCNA monoclonal antibody (dilution 1:100; DAKO Japan Co, Tokyo, Japan) was employed. Briefly, sections were deparaffinized, rehydrated, washed in phosphate-buffered saline (PBS; 3, 5 minutes), and peroxidase activity was quenched using 0.3% H2O2 in methanol for 30 minutes. Subsequently, the samples were washed in PBS and incubated with blocking solution at room temperature for 10 minutes. After rinsing with PBS, sections were incubated with biotinylated mouse anti-PCNA primary antibody in moist chamber for 30 to 60 minutes and then rinsed with PBS. Samples were incubated with streptavidin peroxidase at room temperature for 10 minutes and washed with PBS. The antibodyperoxidase complex was developed using 3,30 -diaminobenzidine chromogen at 18 to 24 C for 2 to 5 minutes. Finally, the sections were washed with PBS, counterstained with hematoxylin for 1 minute, washed with tap water and followed by PBS for 30 seconds, dehydrated through ascending grades of alcohol, delipidated in xylene, and coverslipped with Mount-Quick (Daido Sangyo, Tokyo). Proliferating cell nuclear antigenlabeling index (PCNA-LI). Slides were examined under the light microscope with a magnification of 200 and thin sections were evaluated for PCNA immunostaining. Microscopic fields were chosen at random. Five fields per slide were evaluated. Only the basal germ cells of the seminiferous tubules were counted as the PCNA-LI for each seminiferous tubule and was estimated as a percentage of immunolabeled cells (cells with brown nuclear staining was positive PCNA-ir) to all basal cells according to Tousson et al.16 The average PCNA index in each case was obtained by dividing the sum of all PCNA indices by the number of seminiferous tubules for which the calculation was carried out. For each specimen, the mean + standard error (SE) was calculated.

Animals
The experiment was performed on 32 adult New Zealand rabbits weighing 3.25 + 0.2 kg and of 8 to 9 months of age. The animals were fed pellets standard rabbit ration ad libitum and had free access to water. Animals were divided into 4 groups (8 animals each). Control group (G1) includes animals that were injected intramuscularly with olive oil. Groups 2, 3, and 4 (G2, G3, and G4) include animals that received 1, 2, and 3 intramuscular injections of 5 mg/kg body weight boldenone undecylenate and were dissected after 3, 6, and 9 weeks, respectively.13 At the end of the experiment, the rabbits were fasted for 10 hours and then euthanized with intraperitoneal injection of sodium pentobarbital and subjected to a complete necropsy. Blood samples were individually collected from the inferior vena cava of each rabbit in nonheparinized glass tubes. Blood serum was separated by centrifugation at 3000 rpm for 15 minutes. The collected serum was stored at 18 C. Blood serum was analyzed to determine the concentration of testosterone using radioimmunoassay kit according to the method of Tietz.14

Histological and Immunohistochemical Investigations

The thorax was opened with surgical incision on the sternum and perfusion was done through left ventricle and right atrium. A rinsing solution was perfused before the perfusion of the fixation solution (Bouin fluid). Perfusion with rinsing solution helped overcome this problem. The rinsing solution was prepared by dissolving 9.0 g NaCl, 25 g polyvinyl pyrrolidone, 0.25 g heparin, and 5.0 g Procain-HCL in 1 l of water by thorough stirring. The pH was adjusted to 7.35 with 1 N NaOH and filtered twice through Millipore filters of pore size 3.0 mm or less. The perfusion of both solutions was performed using a scalp vein attached to a 50-cc syringe. Testes were immediately removed, taking care to handle the specimens gently to minimize trauma to the delicate seminiferous tubules prior to placement of each testis into the fixative solution. The tunica albuginea was shallowly pierced at each pole 5 times with a 21-gauge needle to aid in the penetration of the fixative solution. Fixation time was limited to 24 hours and tissues were transferred to 70% ethyl alcohol. Alcohol was changed 3 times daily for 2 days before transferring the specimens to a saturated solution of 70% ethyl alcohol and lithium carbonate to neutralize the picric acid in Bouin fluid. The ethyl alcohollithium carbonate solution was changed 3 or more times until the yellow color of Bouin fluid was almost completely depleted from the tissue. Testes were stored in 70% ethyl alcohol until they were processed. The fixed testis were dehydrated through a graded series of ethanol and embedded in paraffin according to standard procedures. Paraffin sections (5 mm thick) were mounted on gelatin chromalum-coated glass slides and stored

Statistical Analysis
Data were expressed as mean values + SE, and statistical analysis was performed using 1-way analysis of variance to assess the significant differences among the treatment groups. The criterion for statistical significance was set at P < .05 for the biochemical data. All statistical analyses were performed using SPSS statistical version 16 software package (SPSS Inc, Montreal, Canada).

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Table 1. Changes in Live Body Weight, Total Gain, Average Daily Gain, Concentrations of Testosterone Levels and PCNA Index in Testes Tissues in Different Groups Under Studya Live Body Weight (g) Groups G1 G2 G3 G4 Sig. At 0 day (Initial) 3088.3 + 3083.3 + 3186.7 + 3165.0 + NS 127.0 44.1 46.7 55.3 At Final 3833.3 + 124.8 3731.7 + 52.4d 4450.0 + 59.2c 50.91.7 + 68.2b .001
e

Total Gain (g) 745.0 + 5.0 648.3 + 8.8 (21 d)d 1263.3 + 13.6c 1926.7 + 23.3 (63 d)b .000
e

Average Daily 17.9 31.3 30.3 30.7 + 0.06 + 0.67b + 0.33b + 0.33b .000
c

Testosterone Level 0.46 + 0.285 0.66 + 0.384b 1.02 + 0.2459c 1.5 + 0.27d .05
b

PCNA Index 91.3 + 0.3b 67.0 + 1.17c 22.6 + 0.67d 6.7 + 0.33e .05

Abbreviations: PCNA, proliferating cell nuclear antigen; NS, not significant. a Values having different superscript letters within the same column for each factor are significantly different at P < .05.

Results
The animals used as sample appeared healthy and did not show clinical signs of disease and no rabbits died during the experiment. Blood serum analyses of rabbits indicated that BOL injections were significantly increased (P < .05) the testosterone levels in G2, G3, and G4 compared with the control group (Table 1). Table 1 also showed the changes in the body weight rate in different groups under study. The body weight rate significantly increased (P < .05) after treatment with boldenone undecylenate when compared with the control group (Table 1).

sloughing of germ cells into the tubular lumen, and many syncytial cells were present (Figure 1G and H). Immunohistochemical results. Only the spermatogonia in control groups showed a positive strong reaction for PCNA-ir, while the other spermatogenic cell types showed negative reaction (Figure 2A and B). Figure 2B showed a positive reaction for PCNA-ir in some of Sertoli cells in the testes of control rat, which can be identified by their oval- or pear-shaped vesicular nuclei lying perpendicular on the basement membrane of the seminiferous tubules (Figure 2B). Leydig cells of control rats are found in the connective tissue between the seminiferous tubules and showed a negative reaction for PCNA-ir (Figure 2A). The lumen of the seminiferous tubules in the control group was fully packed with sperms that showed a negative reaction for PCNA-ir (Figure 2A). Rabbit testes treated with BOL showed many immunohistochemical changes in testicular tissues (Figure 2C-H). The numbers of spermatogonia that have positive reaction for PCNA-ir were significantly decreased in BOL-treated animals as compared with the control group (Figure 2C-H). Also, the PCNA index values were significantly decreased with increased dose of BOL (Table 1). The Sertoli and Leydig cells in testes showed negative reaction for PCNA-ir after BOL treatment (Figure 2C-G). Some of the spermatogonia found in the lumen of seminiferous tubule showed positive reaction for PCNA-ir (Figure 2E). On the other hand, the syncytial cells in G4 showed negative reaction for PCNA-ir (Figure 2G and H).

Histological Examination
Histopathological study showed that the cycle of spermatogenesis was regular in all male rabbits in the control group (Figure 1A and B). The structural components of the testis are the seminiferous tubules and interstitial tissues (Leydig cells). Two types of cells are identified in rat seminiferous tubules, the Sertoli cells and the spermatogenic cells (spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and sperms). The Sertoli cells, rest on the thin basal lamina (basement membrane), while the spermatogenic cells are arranged in many layers, namely, the spermatogonia, primary and secondary spermatocytes; spermatoids and finally mature spermatozoa (Figure 1B). The histopathological examination of rabbit testes treated with BOL showed various histopathological changes, as with an increase in the dose of BOL (Figure 1C-H). However, the light microscopy examination of rabbit testes treated with BOL showed a significant decrease in the number of spermatogenic cells in the seminiferous tubules (Figure 1D and E). The basement membrane of the seminiferous tubules in G2 had been thickened together with vacuolar degenerative changes in the focal areas in the cytoplasm of the germinal epithelium and in the Sertoli cells (Figure 1D and E). Testicular sections of G3 showed fibrosis, disturbance, and abnormal distribution of spermatocytes in the lumina of the seminiferous tubules (Figure 1E and F). Intertubular spaces and vein congestions increased in the testes of G3 (Figure 1F). Marked morphological changes in the testes of G4 such as degeneration of germinal epithelium,

Discussion
Our results showed that there was a significant increase in the concentration of plasma testosterone after BOL injections in G2, G3, and G4 compared with the control (P < .05). In agreement with our findings, serum testosterone levels in the groups treated with anabolic-androgenic steroid were significantly higher than that in the control group.13,17-19 In contrast, the administration of testosterone alone did not induce any variation in plasma testosterone.20 Also, Shimomura et al21 showed that the treatment of rats with ethinylestradiol alone significantly decreased the testosterone levels in serum and the testis.

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Reproductive Sciences 19(3)

Figure 1. Photomicrographs of rabbit testes stained with hematoxylin and eosin (HE). A and B, Photomicrographs of control rabbit testes showing normal structure of seminiferous tubules (arrows). The cycle of spermatogenesis was regular and the lumen of seminiferous tubules was fully packed with sperms (stars). C and D, Photomicrographs of rabbit testes treated with 1 dose boldenone, showing marked morphological changes such as vacuolation of germinal epithelium and sloughing of germ cells into the tubular lumen. E and F, Photomicrographs of rabbit testes treated with 2 doses of boldenone, showing fibrosis, disturbance, and abnormal distribution of spermatocytes in the lumina of the seminiferous tubules. H and G, Photomicrographs of rabbit testes treated with 3 doses of boldenone, showing degeneration of germinal epithelium, sloughing of germ cells into the tubular lumen in the presence of many syncytial cells (arrows) and reduction of sperms in the seminiferous tubules lumen (star).

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Figure 2. Photomicrographs of proliferating cell nuclear antigen immunoreactivity (PCNA-ir) in the cross sections of rabbit testis. A and B, Numerous numbers of spermatogonia (arrows) and some of Sertoli cells (arrows head) in the seminiferous tubules of control group showing positive reaction of PCNA-ir. C and D, Photomicrographs of rabbit testes treated with 1 dose of boldenone, showing that a small numbers of spermatogonia have positive reaction for PCNA-ir. E and F, Photomicrographs of rabbit testes treated with 2 doses of boldenone, showing that a few numbers of spermatogonia have positive reaction for PCNA-ir (arrows head), also some of spermatogonia in the lumen have positive reaction for PCNA-ir (arrows). G and H, Photomicrographs of rabbit testes treated with 3 doses of boldenone, showing negative reaction for PCNA-ir in spermatogonia and syncytial cells.

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258 The obtained results indicate that intramuscular injection of rabbits with BOL adversely affects spermatogenesis, suggesting that anabolic-androgenic steroid hormone might play an important role not only in controlling normal testicular development but also in maintaining normal testicular function and spermatogenesis. Also, the treated rabbit testes showed marked histological changes in the seminiferous tubules such as thickening of basement membrane together with vacuolar degenerative changes in the focal areas in the cytoplasm of the spermatogenic epithelium, fibrosis, degeneration of germinal epithelium with abnormal distribution of spermatozoa, and many syncytial cells were found in the seminiferous tubule lumen. Our results are in agreement with Groot and Biolatti22 who reported that, BOL induces similar lesions in the testes of cattle. These results are in agreement with Veeramachaneni et al23 who reported that zeranol and estradiol induce similar lesions in the testes and epididymides of the prepubertal beef bull. Also, the present results are in agreement with Silcox et al24 who studied the effects of zeranol and trenbolone acetate on testis function, live weight gain and carcass traits of beef bulls, and with that of Rodriguez et al25 who studied the comparative morphological of lamb and calf Sertoli cells treated with anabolic agent. Animal experiments with anabolic steroids reduced testicular development and increased stromal proliferation.26 Similar findings were reported in lamb and calf testis from animals implanted with estradiol and trenbolone.25 These results are in agreement with a number of recent studies which provided evidence that anabolic steroid causes an adverse effect on the human health.1,2 Our results showed that this histopathological alternation in rabbit testes were increased with an increase in the BOL dose. Yesalis et al27 and Gabr et al13 reported that in addition to the growth-promoting effects, anabolic steroids have been showing to adversely affect the cardiovascular, hepatic, and endocrine systems. Anabolicandrogenic steroids caused some adverse effects on many other adverse effects associated with anabolic-androgenic steroids such as disturbance of the endocrine and immune function, alterations of sebaceous system and skin changes of haemostatic system and urogenital tract.1-3 The seminiferous epithelium contains 2 distinct cell populations, namely somatic Sertoli cells and spermatogenic cells. Spermatogonia undergo successive rounds of mitotic divisions and later the resulting daughter cells enter meiotic division to provide sperm. Proliferating cell nuclear antigen is a wellknown 36-kDa nuclear matrix protein which is essential for multiple cell cycle pathways, including DNA replication, DNA elongation, and DNA excision repair.28 Proliferating cell nuclear antigen is useful for the diagnosis of germinal arrest because the PCNA levels are significantly reduced in germinal arrest, which is an indication of deterioration of DNA synthesis.29 The efficiency of spermatogenesis depends on the proliferative activity of spermatogonia and the loss of germ cells during meiosis and spermiogenesis.30 The present results revealed that of the total number of spermatogonia that showed a positive reaction for PCNA-ir in control and BOL-treated rabbits, was about 91% of spermatogonia in control animals

Reproductive Sciences 19(3) showed a positive reaction for PCNA-ir, while severe reduction in this numbers in BOL-treated rabbits as counted by PCNALI, indicating a germinal arrest in the BOL treated status (defected spermatogenesis). Also, PCNA-ir were detected in Sertoli cells in the testes of control rabbits and not detected in BOL-treated rabbits. The present results indicate that BOL adversely affects spermatogenesis, suggesting that androgenic steroid hormone might play an important role not only in controlling normal testicular development but also in maintaining normal testicular function and spermatogenesis. A severe decrease in the number of spermatogonia was recorded that have positive reaction for PCNA-ir, indicating that sperms in these animals were severely decreased or disappear in the future; this is a marker for infertility.6,16 These findings suggested that the misuse of growth promoter boldenone undecylenate may contribute to a continuous damage to the testicular function and structure, which may lead to infertility. These findings explain the common phenomena in athletics and bodybuilders who suffer from infertility as they were injected with some drugs such as steroids (BOL) to build muscles. Also, these findings suggested that the misuse of growth promoter boldenone undecylenate may contribute to a continuous damage of the testicular function and structure, which may lead to progressive genital diseases, so people should be careful while using such steroids to enhance their strength and endurance. Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.

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