Chemotherapy has improved the quality of life of patients with cancer and given hope for remission. Despite successes, even the most effective anticancer drugs may cause unwanted lesions [1,2]. Cisplatin (CP) (cis-diamminedichloroplatinum-II) is a widely prescribed anticancer drug . It was first approved in 1979 and continues to be used in the treatment of many types of solid tumors, including ovarian, breast, lung, bladder, and head and neck . Moreover, CP is effective in treating testicular cancer, which is the most commonly occurring solid tumor in the male population of the age group 15–35 years as it has a more than 90% cure rate [5,6]. Despite its clinical usefulness, treatment with CP has been associated with several toxic side effects. It has severe deleterious effects on spermatogenesis and patient's fertility, which may be irreversible . The testicular damage following treatment with CP is generally caused by oxidative damage .
There is a lot of interest in several antioxidant agents extracted from plants and dietary components that may reduce some of the side effects of CP without altering the effects of chemotherapy [9,10]. Bee products are frequently used in traditional medicine and have a very long history. Royal jelly (RJ) is a honeybee product secreted from the hypopharyngeal and mandibular glands of worker honeybees. It consists mainly of proteins, sugars, lipids, vitamins, and free amino acids . It has been shown to possess several biological activities, such as antibacterial, anti-inflammatory, antioxidative, immunomodulatory, and tumoricidal activities. It is widely used in commercially available drugs and health foods, as well as in cosmetics, in many countries .
Aim of the work
The aim of the present study was to investigate the effects of CP on testicular structure and plasma testosterone levels in adult albino rats and examine the possible protective effect of RJ on these parameters.
Materials and methods
Twenty-four healthy adult albino rats weighing 220–250g were used in this study. They were housed in standard cages in the Medical Research Center in Ain Shams University. They had free access to water and a standard diet. Animals were kept for 1 week before beginning the experiment for acclimatization.
All animals were randomly divided into four groups of six rats each:
Group I (the control group): The rats in this group received a single intraperitoneal injection of 0.5ml of 0.9% physiologic saline, which was the vehicle for CP.
Group II (the CP group): Rats in this group received a single intraperitoneal injection of CP (Merck Company, Frankfurter Straβe, Darmstadt, Germany) at a dose of 7mg/kg, a dose that is well known to induce testicular toxicity in rats .
Group III (the CP and RJ-treated group): Rats in this group received CP in the same way as did the rats in group II, and then RJ (YS Organic Bee Farms, Sheridan, Illinois,USA) was administered by gavage daily for 10 days at a dose of 100mg/kg .
Group IV (the RJ group): Rats in this group received RJ alone daily at the same previous dose.
At the end of the experiment, all rats were sacrificed by decapitation. Blood samples were collected from all the animals. Each testis was dissected by abdominal incision, cleared from adhering connective tissue, and weighed. The testes were then subjected to the following investigations.
Light microscopic study
One testis from each rat was fixed immediately in aqueous Bouin's solution and processed to obtain paraffin sections of 5μm thickness. They were stained with H&E and caspase-3 to be examined and photographed.
Caspase-3 (ready to use) was used for detection of apoptosis in the germinal epithelium of the seminiferous tubules.
Paraffin sections of the testes and of a positive control (tonsils) were cut at 5 μm thickness on positively charged slides and incubated at 42°C in an oven for 24h. Sections were deparaffinized in xylene (1h), hydrated in descending grades of alcohol, and then incubated in hydrogen peroxide (5min). They were then washed twice in phosphate-buffered saline (5min each). Ultra V block was applied (10min). Ready-to-use primary antibody was applied to the sections, which were then incubated for 1.5h. Thereafter, the sections were washed twice in phosphate-buffered saline for 5min each. Secondary antibody was applied and the sections were again incubated for 20min, following which they were washed three times in phosphate-buffered saline for 5min each. DAB solution was then applied to the sections and they were further incubated for 10min . The sections were then washed in distilled water and counterstained with Mayer's hematoxylin (2min), following which they were washed in tap water, dehydrated, cleared, and mounted by DPX.
Negative controls were processed according to the same protocol, except for the use of the primary antibody.
Electron microscopic study
The other testis from each rat was decapsulated and the exposed dispersed seminiferous tubules were cut into small pieces of about 1mm3 size and rapidly fixed in 2.5% glutaraldehyde for 24h. Specimens were washed in 0.1mol/l phosphate buffer at 4°C and then postfixed in 1% osmium tetroxide at room temperature. Specimens were dehydrated in ascending grades of ethyl alcohol and embedded in Epon resin, Momentive Specialty Chemicals Epoxy Resins (Houston, TX) USA. Semithin sections of 1μm were stained with 1% toluidine blue in borax and examined under a light microscope. Ultrathin sections of 50nm were cut, mounted on copper grids, and stained with uranyl acetate and lead citrate . Specimens were examined and photographed using a JEM 1200 EXII, JEOL, Tokyo, Japan transmission electron microscope (TEM) at the Faculty of Science, Ain Shams University.
Morphometric and statistical study
Five fields from three H&E-stained sections of each rat from each group were examined to measure the mean diameter and the mean germinal epithelium thickness of the seminiferous tubules using an image analyzer (Olympus Image J, NIH, 1.41b, America Inc, Melville, NY, USA) at the Oral Pathology Department, Faculty of Dentistry, Ain Shams University. Rounded transversely cut tubules were selected and two diameters of each tubule, one perpendicular to the other, were measured and the average taken . When the sections were oblique, only the minor axis was considered for the measurements [17,18]. The mean diameter and the mean thickness of the germinal epithelium of the seminiferous tubules, as well as the mean weight of the testes and the mean blood testosterone level of the animals at the time of scarification, were measured. All values were presented as mean ± SD. The differences among the groups with respect to all measured data parameters were statistically analyzed using one-way analysis of variance and the post-hoc test using SSPS program version 17 (IBM Corporation, Somers, New York, USA). The calculations were considered significant if P value was less than 0.05.
Light microscopic results
Examination of sections from group I stained by H&E showed closely packed seminiferous tubules separated by narrow interstitial spaces containing clusters of interstitial Leydig cells with vesicular nuclei. Each seminiferous tubule was ensheathed by a well-defined basement membrane surrounded by flattened myoid cells with flattened nuclei. The tubules were lined with stratified germinal epithelium formed of spermatogonia, primary spermatocytes, and early and late spermatids. Sertoli cells with large pale nuclei were seen between the spermatogenic cells, resting on the basement membrane. Spermatozoa were also seen within the luminal compartment (Fig. 1). Toluidine blue-stained semithin sections of the control group revealed Sertoli cells with large oval nuclei with prominent nucleoli and spermatogonia with oval nuclei resting on a regular basement membrane. Primary spermatocytes appeared as the largest spermatogenic cells. Several layers of early spermatids were seen with their central rounded nuclei. Late spermatids were identified by their elongated deeply stained nuclei (Fig. 2). Caspase-3-stained sections of the same group showed apparently normal spermatogenic cells with negative caspase-3 reaction (Fig. 3).
The H&E-stained sections of group II (which received CP alone) showed widening of the interstitial spaces between the seminiferous tubules, which were occupied by areas of extensive homogenous acidophilic exudates and congested thick-walled blood vessels (Fig. 4). A few seminiferous tubules were noticed with apparently normal spermatogenic cells (Fig. 5). Others appeared severely depleted with extensive loss of their germinal epithelium (Figs 5 and 6). Extensive epithelial vacuolations were also noticed in these tubules (Fig. 6). Other tubules were seen partially depleted from their spermatogenic cells and were seen resting on a corrugated basement membrane. Wide separation between the epithelium and the basement membrane was also noticed in some areas (Fig. 7). Toluidine blue-stained semithin sections of the same group showed the wall of some seminiferous tubules formed mainly of Sertoli cells with vacuolated cytoplasm. Moreover, a few spermatogenic cells were noticed with large areas of cellular loss (Fig. 8). Other seminiferous tubules were noticed to be formed of disorganized germinal epithelial layers resting on an irregular basement membrane with darkly stained Sertoli cells. Wide intercellular vacuoles and cytoplasmic vacuoles were also seen in the germinal epithelium (Fig. 9). Sections of the same group stained by caspase-3 showed multiple apoptotic germinal cells with positive caspase-3 reaction (Fig. 10).
Examination of sections from group III (the CP group treated with RJ) stained by H&E showed that most seminiferous tubules were nearly similar to those of the control group. They were lined by several layers of spermatogenic cells resting on a regular basement membrane. Interstitial spaces were apparently narrow with few areas of acidophilic exudates (Figs 11 and 12), whereas in some tubules wide intercellular spaces were seen between spermatogenic cells (Fig. 12). Toluidine blue-stained semithin sections of the same group showed seminiferous tubules with their lining of spermatogenic epithelium nearly similar to that of the control group, although wide intercellular spaces were seen between spermatogenic cells in a few areas (Fig. 13). Sections of the same group stained by caspase-3 showed some apoptotic germinal cells with positive caspase-3 reactions especially in the spermatid stage (Fig. 14).
Examination of different sections from group IV (which received RJ alone) revealed features nearly similar to those of the control group (Fig. 15).
Electron microscopic results
The control group (group I) showed dome-shaped spermatogonia with oval nuclei and Sertoli cells with large pale euchromatic indented nuclei. Their cytoplasm contained many smooth endoplasmic reticulum (sER), mitochondria, and lipid droplets. Both cells appeared to be resting on a regular basement membrane surrounded by myoid cells with flattened nuclei. Early spermatids were detected by many peripherally arranged mitochondria with a clear matrix (Fig. 16). Late spermatids were seen with their acrosomal cap covering the anterior half of the nuclei. Microtubules were seen arising from the posterior margin of the acrosomal cap forming the manchette (Fig. 17). Interstitial Leydig cells were noticed having large euchromatic nuclei with a rim of peripheral dense chromatin, abundant sER, mitochondria, and electron-dense bodies of variable size (Fig. 18).
Group II showed Sertoli cells with small dark nuclei resting on a thickened, irregular basement membrane. Their cytoplasm appeared vacuolated, and late spermatids were seen attached to their apices (Fig. 19). Spermatids were noticed with their acrosomal cap covering the anterior half of the irregular nuclei. Cytoplasmic vacuoles were seen surrounding the head cap and dispersed in the cytoplasm, and mitochondria were seen distributed throughout the cell cytoplasm. Prominent Golgi complexes were also noticed (Fig. 20). Leydig cells appeared with apparently abnormal eccentric nuclei. Their cytoplasm showed dilated sER and numerous variable sizes of electron-dense bodies compared with those of the control group (Fig. 21).
Group III showed Sertoli cells with large indented nuclei and apparent nucleoli. The cells were seen resting on a more regular basement membrane, compared with group II. Few cytoplasmic vacuolations were seen in the Sertoli cells and spermatogenic cells. Spermatogonia, primary spermatocytes, and early spermatids were also noticed and were nearly identical to those in the control group (Figs 22 and 23). Cytoplasmic vacuoles were seen in a few late spermatids (Fig. 23). Leydig cells appeared with euchromatic nuclei containing peripheral heterochromatin, many mitochondria, lipid droplets, and lipochrome pigments of variable size (Fig. 24).
Examination of different sections of group IV revealed features that were nearly similar to those of the control group.
Morphometric and statistical results
Weight of the testis
Table 1 and Histogram 1 show the mean ± SD of the weight of the testes in different groups. Group II (the CP group) showed a significant (P < 0.05) decrease in the mean weight of the testes compared with the other groups. In contrast, group III (the RJ-treated group) showed a significant (P < 0.05) increase compared with group II, whereas a nonsignificant (P > 0.05) change was noticed when compared with the control group and group IV.
Diameter of the seminiferous tubules
Table 2 and Histogram 2A show the mean ± SD of the diameter of the seminiferous tubules in different groups. Group II (the CP group) showed a significant (P < 0.05) decrease in the diameter of the seminiferous tubules compared with the other groups. In contrast, group III (the RJ-treated group) showed a significant (P < 0.05) increase in the mean diameter of the seminiferous tubules compared with group II, whereas a nonsignificant (P > 0.05) change was noticed when compared with the control group and group IV.
Thickness of the germinal layer in the seminiferous tubules
Table 2 and Histogram 2B show the mean ± SD of the mean thickness of the germinal layer in the seminiferous tubules of different groups. Group II (the CP group) showed a significant (P < 0.05) decrease in the mean thickness of the seminiferous epithelium compared with the other groups. In contrast, group III (the RJ-treated group) showed a significant (P < 0.05) increase in the mean thickness of the seminiferous epithelium compared with group II, whereas a nonsignificant (P < 0.05) change was noticed when compared with the control group and group IV.
Table 3 and Histogram 3 show the mean ± SD testosterone level in different groups. Group II showed a significant decrease (P < 0.05) in plasma testosterone level compared with the other groups, whereas group III (the RJ-treated group) showed a significantly (P < 0.05) increased plasma testosterone level compared with the CP group (group II). However, the plasma testosterone level in group III was still significantly (P < 0.05) reduced compared with the control group and group IV. Meanwhile, group IV (the RJ group) showed a significant (P < 0.05) increase in testosterone level compared with the other groups.
CP-based chemotherapy results in damage to different tissues, one of them being the testes [1,7,19,20]. Some investigators  reported that CP administration caused temporary or permanent azoospermia or oligospermia. The results of the present study revealed that administration of 7mg/kg of CP to rats resulted in degeneration of the germinal epithelium of some seminiferous tubules. Similar findings were reported by other researchers who described it as a ‘washed out’ appearance, reflecting the extensive cell loss that occurred after exposure to CP . These findings were confirmed by the current histomorphometric results, as a significant decrease in the diameter of the seminiferous tubules and a significant decrease in the height of their germinal epithelium were noticed in the CP group when compared with the control group. Similar findings were also observed by other researchers [3,8,23] who noticed atrophy of the germinal epithelium with administration of CP, which might be the reason for the observed statistically significant decrease in the weight of the testes in the current study in rats treated with CP when compared with the control group. This was in accordance with the observations made by other researchers [3,24]. Regarding the mechanism of action of CP, it was reported that the observed testicular damage following CP exposure could be attributed to the oxidative damage. Oxidative stress is a condition that is associated with an imbalance between the production and removal of reactive oxygen species (ROS) and free radicals . Treatment with CP led to lipid peroxidation and decreased the activity of enzymes that protect against oxidative damage in the testes . Many ROS were also generated by CP, which could interfere with the antioxidant defense system . Excess ROS generation had damaging effects on various cell components .
In the current study, in the CP group, spermatogenic cells were seen to be widely separated from each other and were observed resting on a corrugated basement membrane. Moreover, widened interstitial spaces between the seminiferous tubules were occupied by areas of exudates. Congested thick-walled blood vessels were also observed. Similar findings were also seen by other researchers [8,23]. Moreover, it was reported that a decrease in testosterone production might occur after CP treatment and might directly or indirectly affect the interstitial tissue and lymphatic space, provoking interstitial testicular edema . It was also reported that capillary filtration participates in interstitial fluid formation; thus, changes in capillary permeability and blood/lymph flow could modify the interstitial fluid . Alterations in the composition or volume of the interstitial fluid could also significantly affect the testicular function .
In the current study, the walls of most seminiferous tubules were seen to be mainly formed of spermatogenic cells and Sertoli cells, which appeared with vacuolated cytoplasm and darkly stained nuclei. Similarly, it was noticed that, with administration of CP, most tubules contained only the early stages of differentiating germ cells . They attributed this finding to the uninterrupted replenishment of germ cells by the mitotic divisions of spermatogonial stem cells. Meanwhile, some investigators observed a significant damage to Sertoli and germ cell populations as a result of CP administration [20,30]. Treatment of mice with 10 mg/kg (8 days) of CP resulted in the formation of unusually large empty lumens of the seminiferous tubules that were devoid of round and mature spermatids .
In the present work multiple apoptotic germinal cells were noticed in the CP group with positive caspase-3 reaction. Some researchers monitored apoptosis within the first 72 h after CP injection . They noticed the appearance of apoptosis in spermatocytes before any evidence of damage to other cell types in the seminiferous tubules became visible. Similarly, previous studies  reported that, whereas spermatocytes and spermatids were vulnerable to the toxic effects of modest CP doses, the stem cell spermatogonia were not. It was suggested that a direct effect such as germ cell DNA damage might trigger the apoptotic response in the germ cells [5,8]. In addition, it was reported that CP affects spermatogenesis by inhibiting the nucleic acid synthesis of germ cells . Meanwhile, Meistrich et al.  stated that injured Sertoli cells affected germ cells through paracrine signaling to undergo apoptosis.
In the current study, most Leydig cells by TEM examination appeared with an apparently abnormal eccentric nuclei, and their cytoplasm showed dilated sER, numerous lipochrome pigments, and lipid droplets. Similar findings were reported by other researchers . They noticed impairment of Leydig cells following chemotherapy, but they reported that the mechanism of this impairment was not well known. They reported that chemotherapy might have a direct toxic effect on Leydig cells, but there was also germinal epithelial damage that might indirectly affect the Leydig cell function.
In the current study, it was found that the blood testosterone level in the CP group was significantly decreased compared with the control group. Similarly, it was reported that CP could decline the testosterone levels , which might be related to Leydig cell morphological alterations . This was against findings by other researchers who reported that the testosterone level in the CP group was not significantly different from that of the control group .
In the present work, most CP-induced changes in the testes were reversed by treatment with RJ. Most seminiferous tubules were seen to be almost similar to those of the control group. They were lined by several layers of spermatogenic cells resting on a regular basement membrane. These findings were confirmed by the current histomorphometric results. Significant increase in the diameter of the seminiferous tubules and a significant increase in the thickness of their germinal epithelium were noticed when compared with the CP group. In the current study the interstitial spaces between the seminiferous tubules were apparently narrower in the RJ-treated group compared with the CP group. Moreover, few areas of acidophilic exudates were seen in these spaces. In some tubules, wide intercellular spaces were seen between spermatogenic cells. Few apoptotic germinal cells were still seen with positive caspase-3 reactions, especially in the spermatid stage. With TEM examination, cytoplasmic vacuolations were observed in some Sertoli cells and spermatogenic cells. Leydig cells appeared almost similar to those seen in controls.
Regarding the plasma testosterone level in the RJ-treated group, there was a significant increase when compared with the CP group; however, the level was still significantly reduced compared with the control group. A significant increase was noticed in the weights of the testes in the RJ-treated group when compared with the CP group. In the current study, administration of RJ alone to normal rats resulted in a profile close to that of the control group with respect to histopathology and testis weight. In the present work, the mean diameter of the seminiferous tubules and their mean wall thickness were similar to those of control rats. However, a significant increase was noticed in the blood testosterone level of the RJ group when compared with the other groups. This finding coincided with the findings of some researchers  who reported that RJ contains bioactive substances including reproductive system development-stimulating factor, which might stimulate testosterone release. In contrast, Atessahin et al.  noticed that with administration of RJ alone the testosterone levels were close to those in the control group.
Several studies have been conducted on the immunomodulatory mechanism of natural substances that could modulate the state of immunodeficiency that occurs with cancer. RJ had an immunomodulatory effect . It was also demonstrated that the protein fractions in the RJ had a high antioxidative activity and scavenging ability against free radicals . Hence, RJ with antioxidant and immunomodulatory activities might be useful in the prevention of side effects of CP-induced sperm toxicity . Moreover, some investigators  reported that RJ contains biologically active amino acids such as proline, cystine, and cysteine. Proline protects membranes and proteins from various imposed stress conditions and it might also act as an antioxidant agent , whereas cystine and cysteine participate in the synthesis of glutathione, which is an effective cellular antioxidant that breaks down ROS and detoxifies carcinogens .
Conclusion and recommendation
From the current study it was concluded that RJ had a protective role against CP-induced testicular injury. Hence, it is recommended for patients who receive CP to take RJ during their course of treatment.
Table. No title avai...Image Tools
Conflicts of interest
There are no conflicts of interest.
Tohamy AA, El Ghor AA, El Nahas SM, Noshy MM. Beta-glucan inhibits the genotoxicity of cyclophosphamide, adriamycin and cisplatin. Mutat Res. 2003;541:45–53
Weijl NI, Elsendoorn TJ, Lentjes EGWM, Hopman GD, Wipkink Bakker A, Zwinderman AH, et al. Supplementation with antioxidant micronutrients and chemotherapy-induced toxicity in cancer patients treated with cisplatin-based chemotherapy: a randomised, double-blind, placebo-controlled study. Eur J Cancer. 2004;40:1713–1723
Atessahin A, Karahan I, Türk G, Gür S, Yilmaz S, Çeribasi AO. Protective role of lycopene on cisplatin-induced changes in sperm characteristics, testicular damage and oxidative stress in rats. Reprod Toxicol. 2006;21:42–47
Elwell KE, Hall C, Tharkar S, Giraud Y, Bennett B, Bae C, Carper SW. A fluorine containing bipyridine cisplatin analog is more effective than cisplatin at inducing apoptosis in cancer cell lines. Bioorg Med Chem. 2006;14:8692–8700
Wang D, Lippard SJ. Cellular processing of platinum anticancer drugs. Nat Rev Drug Discov. 2005;4:307–320
Aminsharifi AR, Talaei T, Kumar V, Sabayan B, Samani S, Mohamadhoseini E. A postulated role of testosterone for prevention of cisplatin gonadal toxicity. Med Hypotheses. 2007;68:525–527
Cherry SM, Hunt PA, Hassold TJ. Cisplatin disrupts mammalian spermatogenesis, but does not affect recombination or chromosome segregation. Mutat Res. 2004;564:115–128
Ilbey YO, Ozbek E, Cekmen M, Simsek A, Otunctemur A, Somay A. Protective effect of curcumin in cisplatin-induced oxidative injury in rat testis: mitogen-activated protein kinase and nuclear factor-kappa B signaling pathways. Hum Reprod. 2009;24:1717–1725
Pérez Rojas JM, Cruz C, García López P, Sánchez González DJ, Martínez Martínez CM, Ceballos G, et al. Renoprotection by α-mangostin is related to the attenuation in renal oxidative/nitrosative stress induced by cisplatin nephrotoxicity. Free Radic Res. 2009;43:1122–1132
Sánchez Pérez Y, Morales Bárcenas R, García Cuellar CM, López Marure R, Calderon Oliver M, Pedraza Chaverri J, Chirino YI. The alpha-mangostin prevention on cisplatin-induced apoptotic death in LLC-PK1 cells is associated to an inhibition of ROS production and p53 induction. Chem Biol Interact. 2010;188:144–150
Takenaka T. Chemical composition of royal jelly. Honeybee Sci. 1982;3:69–74
Nakajima Y, Tsuruma K, Shimazawa M, Mishima S, Hara H. Comparison of bee products based on assays of antioxidant capacities. BMC Complement Altern Med. 2009;9:4
Yapar K, Çavusoglu K, Oruç E, Yalçin E. Protective effect of royal jelly and green tea extracts effect against cisplatin-induced nephrotoxicity in mice: a comparative study. J Med Food. 2009;12:1136–1142
Bancroft J, Gamble M Theory and practice of histological techniques. 20025th ed. Philadelphia Churchill Livingstone
Bozzola JJ, Russell LD Electron microscopy: principles and techniques for biologists. 19992nd ed. Sudbury, MA, Canada, UK, library of congress Jones & Bartlett
Ayana K, Prashanthi N, Nayanatara A, Bairy LK, D'Souza UJA. An organophosphate insecticide methyl parathion (O-O-dimethyl O-4-nitrophenyl phosphorothioate) induces cytotoxic damage and tubular atrophy in the testis despite elevated testosterone level in the rat. J Toxicol Sci. 2006;31:177–189
Stumpp T, Sasso Cerri E, Freymüller E, Miraglia SM. Apoptosis and testicular alterations in albino rats treated with etoposide during the prepubertal phase. Anat Rec A Discov Mol Cell Evol Biol. 2004;279:611–622
Lirdi LC, Stumpp T, Sasso Cerri E, Miraglia SM. Amifostine protective effect on cisplatin-treated rat testis. Anat Rec. 2008;291:797–808
Ishikawa T, Kamidono S, Fujisawa M. Fertility after high-dose chemotherapy for testicular cancer. Urology. 2004;63:137–140
Pectasides D, Pectasides M, Farmakis D, Nikolaou M, Koumpou M, Kostopoulou V, Mylonakis N. Testicular function in patients with testicular cancer treated with bleomycin-etoposide-carboplatin (BEC(90)) combination chemotherapy. Eur Urol. 2004;45:187–193
Howell SJ, Shalet SM. Testicular function following chemotherapy. Hum Reprod Update. 2001;7:363–369
Seaman F, Sawhney P, Giammona CJ, Richburg JH. Cisplatin-induced pulse of germ cell apoptosis precedes long-term elevated apoptotic rates in C57/BL/6 mouse testis. Apoptosis. 2003;8:101–108
Ilbey YO, Ozbek E, Simsek A, Cekmen M, Otunctemur A, Somay A. Chemoprotective effect of a nuclear factor-κB inhibitor, pyrrolidine dithiocarbamate, against cisplatin-induced testicular damage in rats. J Androl. 2009;30:505–514
Türk G, Atessahin A, Sönmez M, Ceribasi AO, Yüce A. Improvement of cisplatin-induced injuries to sperm quality, the oxidant-antioxidant system and the histologic structure of the rat testis by ellagic acid. Fertil Steril. 2008;89(Suppl):1474–1481
Baker MA, Aitken RJ. Reactive oxygen species in spermatozoa: methods for monitoring and significance for the origins of genetic disease and infertility. Reprod Biol Endocrinol. 2005;3:67
Badary OA, Abdel Maksoud S, Ahmed WA, Owieda GH. Naringenin attenuates cisplatin nephrotoxicity in rats. Life Sci. 2005;76:2125–2135
Longo V, Gervasi PG, Lubrano V. Cisplatin induced toxicity in rat tissues: the protective effect of Lisosan G. Food Chem Toxicol. 2011;49:233–237
Aydiner A, Aytekin Y, Topuz E. Effects of cisplatin on testicular tissue and the Leydig cell-pituitary axis. Oncology. 1997;54:74–78
Maddocks S, Setchell BP. The composition of extracellular interstitial fluid collected with a push-pull cannula from the testes of adult rats. J Physiol. 1988;407:363–372
Sanocka D, Kurpisz M. Reactive oxygen species and sperm cells. Reprod Biol Endocrinol. 2004;2:12
Vawda AI, Davies AG. Effects of cisplatin on the mouse testis. Acta Endocrinol. 1986;112:436–441
Adler ID, El Tarras A. Clastogenic effects of cis-diamminedichloroplatinum. II. Induction of chromosomal aberrations in primary spermatocytes and spermatogonial stem cells of mice. Mutat Res. 1990;243:173–178
Meistrich ML, Chawla SP, da Cunha MF, Johnson SL, Plager C, Papadopoulos NE, et al. Recovery of sperm production after chemotherapy for osteosarcoma. Cancer. 1989;63:2115–2123
Colpi GM, Contalbi GF, Nerva F, Sagone P, Piediferro G. Testicular function following chemo-radiotherapy. Eur J Obstet Gynecol Reprod Biol. 2004;113(Suppl 1):S2–S6
Malarvizhi D, Mathur PP. Effects of cisplatin on testicular functions in rats. Indian J Exp Biol. 1996;34:995–998
Huang HF, Pogach LM, Nathan E, Giglio W. Acute and chronic effects of cisplatinum upon testicular function in the rat. J Androl. 1990;11:436–445
Nagai T, Inoue R. Preparation and the functional properties of water extract and alkaline extract of royal jelly. Food Chem. 2004;84:181–186
Sver L, Oršolic N, Tadic Z, Njari B, Valpotic I, Bašic I. A royal jelly as a new potential immunomodulator in rats and mice. Comp Immunol Microbiol Infect Dis. 1996;19:31–38
Seminotti B, Leipnitz G, Amaral AU, Fernandes CG, da Silva Lde B, Tonin AM, et al. Lysine induces lipid and protein damage and decreases reduced glutathione concentrations in brain of young rats. Int J Dev Neurosci. 2008;26:693–698
Parodi PW. A role for milk proteins and their peptides in cancer prevention. Curr Pharm Des. 2007;13:813–828