Protective Role of Royal Jelly in Oxymetholone-induced Oxidative Injury in Mouse Testis

Abstract

ABSTRACT
Background: An adverse effect of oxymetholone (OXM), an anabolic-androgenic steroid used as energetic medicine, is reproductive toxicity. Royal jelly (RJ) is an efficient antioxidant that has been used to treat reproductive problems. In this study, we investigated the effects of RJ on OXM-induced oxidative injuries in mouse testes.
Methods: Male mice were divided into four groups. Two groups of mice were administered OXM (5 mg/kg/day, p.o.) for 28 days. One of these groups received RJ (100 mg/kg/day, p.o.) concurrently. A vehicle-treated control group and a RJ control group were also included.
Results: The OXM-treated group showed a significant decrease in the serum testosterone concentration and spermatogenic activities, along with many histological alterations. OXM treatment also caused a significant decrease in catalase activity with an increase in lipid peroxidation in the mouse testes. The above-noted parameters were restored to near normal levels by RJ co-administration.
Conclusion: The results demonstrate that RJ protects against OXM-induced reproductive toxicities.
Keywords: Mouse, Oxymetholone, Royal Jelly, Testis.

Full text

Iranian Journal of Toxicology Volume 8, No 25, Summer 2014
1. Department of Basic Sciences, Urmia University, Urmia, Iran.
2. Department of Biology, Urmia University, Urmia, Iran.
*Corresponding Author: E-mail: a.shalizar@urmia.ac.ir
Protective Role of Royal Jelly in Oxymetholone-induced Oxidative
Injury in Mouse Testis
Gholamreza Najafi1, Vahid Nejati2, Ali Shalizar Jalali*1, Ensieh Zahmatkesh2
Received: 01.10.2013 Accepted: 30.11.2013
ABSTRACT
Background: An adverse effect of oxymetholone (OXM), an anabolic-androgenic steroid
used as energetic medicine, is reproductive toxicity. Royal jelly (RJ) is an efficient
antioxidant that has been used to treat reproductive problems. In this study, we
investigated the effects of RJ on OXM-induced oxidative injuries in mouse testes.
Methods: Male mice were divided into four groups. Two groups of mice were
administered OXM (5 mg/kg/day, p.o.) for 28 days. One of these groups received RJ (100
mg/kg/day, p.o.) concurrently. A vehicle-treated control group and a RJ control group were
also included.
Results: The OXM-treated group showed a significant decrease in the serum
testosterone concentration and spermatogenic activities, along with many histological
alterations. OXM treatment also caused a significant decrease in catalase activity with an
increase in lipid peroxidation in the mouse testes. The above-noted parameters were
restored to near normal levels by RJ co-administration.
Conclusion: The results demonstrate that RJ protects against OXM-induced
reproductive toxicities.
Keywords: Mouse, Oxymetholone, Royal Jelly, Testis.
IJT 2014; 1073-1080
INTRODUCTION
Worldwide, up to 20% of couples are
infertile. Approximately 30-50% of human
infertility is attributable to male infertility [1,
2]. Although hormonal causes of male
infertility are well known, lack of sufficient
knowledge about intracellular mechanisms
leading to the production of important factors
necessary for regulating spermatogenesis is
the main reason behind the inability to
diagnose and treat certain forms of idiopathic
infertility [3]. Testis, the main organ of the
male reproductive system, consists of two
parts: the testis parenchyma and intercellular
substances. The parenchyma is composed of
long and coiled tubes called seminiferous
tubules. As the seminiferous tubules comprise
approximately 80% of the testicular mass, the
morphological measurements of seminiferous
tubules are important in the studies of testis
tissue [4-7].
Anabolic-androgenic steroids (AASs)
are used by a considerable proportion of the
community to enhance their physique and
performance. More than one million of
Americans use or have used AASs [8].
Oxymetholone (OXM) is an active nutritional
17α-alkylated anabolic-androgenic steroid
derived from testosterone [9]. Since OXM
can stimulate erythropoiesis and increases
erythropoietin production, it is used for the
treatment of anemias caused by low red cell
production [10]. In addition, this drug is
currently applied to the treatment of
myelotoxic anticancer drugs-induced
myelofibrosis [9] and AIDS-associated
wasting [11]. Despite its therapeutic
significance, it has been reported that OXM
causes hepatic and cardiac damages [12, 13]
as well as reproductive toxicities in humans
and experimental animals [14-16]. Also,
AASs have been found to induce
hypogonadotrophic hypogonadism via
negative feedback to the hypothalamus
leading to testicular atrophy and impaired
spermatogenesis [17, 18].

Iranian Journal of Toxicology Gholamreza Najafi et al
1074 Volume 8, No 25, Summer 2014; http://www.ijt.ir
Royal jelly (RJ), a secretion product of
the hypopharyngeal and mandibular glands of
nurse bees, is a mixture that contains many
important compounds with biological activity
such as free amino acids, proteins, sugars,
fatty acids, minerals, and vitamins [19]. Due
to its complex composition, RJ possesses
numerous pharmacological properties
including antioxidant, anti-inflammatory,
antitumor, anti-allergic, antibiotic,
hypotensive, neurotrophic, and
immunomodulatory activities [20-23].
Furthermore, independent studies have
indicated that RJ has positive effects on the
reproductive system and fertility in humans
and animals [24-27]. Hence, the present study
was designed to investigate the probable
protective effects of RJ on OXM-induced
oxidative injuries in mouse testes.
MATERIALS AND METHODS
Animals
This study was carried out on healthy
adult sexually mature male (9 weeks of age)
mice weighing 30±2 g. A total of 32 mice
were obtained from the Animal House of
Faculty of Science, Urmia University, Urmia,
Iran. The animals were housed in filter-top
polycarbonate cages in an air-conditioned
room (temperature: 25±2 ◦C, relative
humidity: 50±10%, and 12 h light/12 h dark
photoperiod) free from any sources of
chemical contamination with free access to
standard diet and water throughout the
experimental period. The experimental
protocol and procedures used in this study
were approved by the Ethics committee of the
Urmia University, Urmia, Iran for the care
and use of laboratory animals.
Experimental design
After seven days of acclimation to the
environment, the mice were randomly divided
into four treatment groups of eight animals
each (n = 8) and treated orally for 28 days as
follows:
Group I (Control): received saline vehicle
(0.1 ml/mice)
Group II (OXM): OXM (5 mg/kg)
Group III (RJ): royal jelly (100 mg/kg)
Group IV (OXM + RJ): royal jelly (100
mg/kg) + OXM (5 mg/kg).
Sampling
Animals were euthanized by CO2
exposure in a special device following
anesthesia with ketamine (75 mg/ kg, IP) 24
hours after the last treatment. Blood was
collected from atrium in routine biochemical
test tubes without anticoagulant for
serological analysis. The abdominal cavity
was opened up through a midline abdominal
incision and the testes were excised quickly.
One half of the right and left testes were fixed
in Bouin’s fixative (0.2% picric acid/2%
(V/V) formaldehyde in PBS) for histological
evaluation and the other halves were kept
frozen at -70 °C until they were homogenized
for further biochemical studies.
Assessment of catalase activity
Catalase (CAT) activity in
homogenized testicular tissue was determined
according to Aebi (1984) [28]. Its activity was
assayed by determining the rate of
degradation of hydrogen peroxide at 240 nm
in 10 mM of potassium phosphate buffer (pH
7.0). Extinction coefficient of 43.6 mM/cm
was used for calculation. One unit is defined
as 1 pmol of hydrogen peroxide consumed
per minute, and the specific activity is
reported as units/mg of protein.
Assessment of lipid peroxidation
Lipid peroxidation (LPO) was
determined by the spectrophotometric TBA
assay as previously described [29].
Malondialdehyde (MDA), formed as an end
product of the peroxidation of lipids, reacts
with thiobarbituric acid (TBA) to generate a
colored product that can be measured
optically at 532 nm. Results were expressed
as µmol MDA/mg protein.
Testosterone assessment
Serum concentration of testosterone
was measured by enzyme-linked
immunosorbent assay (ELISA) as described
in the instructions provided by manufacturer’s
kit (Demeditec Diagnostics GmbH,
Germany).
Histological analysis
After fixation of testes, they were
dehydrated through a gradual series of alcohol

Protective Role of Royal Jelly in Oxymetholone… Iranian Journal of Toxicology
1075
http://www.ijt.ir; Volume 8, No 25, Summer 2014
and cleared in three changes of xylene before
embedded in paraffin. Thin sections (5µm)
perpendicular to the longest axis of the testis
were cut using a microtome and stained with
hematoxylin and eosin according to the
standard method. Histological analysis was
performed under light microscope in terms of
the changes in different groups as compared
to the control group.
Determination of histological parameters
For each testis, five vertical sections
from the polar and the equatorial regions were
sampled [30] and an unbiased numerical
estimation of the following histological
parameters was determined using a systematic
random scheme.
Seminiferous tubules diameter (STsD)
and interstitial tissue thickness (ITT): For
measuring STsD and ITT, 200 round or
nearly round cross-sections of seminiferous
tubules were randomly analyzed in each
mouse (one hundred per testis). Then, two
perpendicular diameters of each cross-section
of seminiferous tubules were measured using
an ocular micrometer of light microscopy
(Olympus Co., Germany) and their means
were calculated. Also, ITT was measured in 4
equidistance of each cross-section of
seminiferous tubules and their means were
calculated [31].
Number of mononuclear immune cells
(MNICs) in testicular tissue: The number of
MNICs was determined by using the unbiased
counting frame proposed by Gundersen
(1977) [32].
Tubule differentiation index (TDI) and
spermiation index (SPI): 200 cross-sections of
seminiferous tubules were randomly analyzed
in each mouse (one hundred per testis) for the
calculation of TDI and SPI. TDI is the
percentage of seminiferous tubules containing
at least three differentiated germ cells [33].
SPI is the percentage of seminiferous tubules
with normal spermiation [34].
Sertoli cell index (SCI) and mitotic
index (MI): Sixty seminiferous tubules per
group were randomly examined for the
calculation of SCI and MI. SCI is the ratio of
the number of germ cells to the number of
Sertoli cells identified by a characteristic
nucleus and nucleolus in all seminiferous
tubules [35]. MI, the number of round
spermatids for each pachytene primary
spermatocytes, was calculated for
determination of cell loss percentage during
cell division [36].
Statistical analysis
The results are expressed as the mean ±
standard error of mean (S.E.M.). Differences
between the groups were assessed by the
analysis of variance (ANOVA) using SPSS
software package for Windows. Statistical
significance between groups was determined
by Tukey’s multiple comparison post hoc test
and the P-values less than 0.05 were
considered to be statistically significant.
RESULTS
Antioxidant status study
The effects of different treatments on
CAT activity and MDA level in testis are
depicted in Table 1. Treatment with OXM
alone resulted in a significant decrease in
CAT activity in testis tissue, whereas it
caused a significant increase in MDA in the
same tissue. Treatment with RJ in
combination with OXM resulted in a
significant improvement in these parameters
in testis tissues compared to the OXM alone
group (Table 1).
Table 1. Effect of oxymetholone and Royal jelly on the testis antioxidant status.
Control OXM RJ OXM+RJ
CAT (U/mg pt.)
51.6±0.6 23.1±0.2
a
53.4±0.5
b
32.3±0.3
a,b
MDA (µmol/mg pt.) 3.9±0.1 6.2±0.2
a
3.8±0.2
b
4.7±0.1
a,b
The values are expressed as mean ± S.E.M. (n = 8).
a Significant differences as compared with the control group at P <0.05
b Significant differences as compared with the OXM group at P <0.05

Iranian Journal of Toxicology Gholamreza Najafi et al
1076 Volume 8, No 25, Summer 2014; http://www.ijt.ir
Testosterone level
Results of testosterone concentrations
study (Figure 1) revealed that treatment with
OXM alone caused a significant decrease in
serum testosterone level as compared to the
control group. The administration of RJ along
with OXM significantly restored serum
testosterone level towards the control value
(Figure 1).
Figure 1. Effect of oxymetholone and Royal jelly on serum concentrations of testosterone.
The values are expressed as mean ± S.E.M. (n =8).
a Significant differences as compared with the control group at P <0.05
b Significant differences as compared with the oxymetholone group at P <0.05
Histological parameters
As seen in Table 2, treatment of male
mice with OXM caused a significant decrease
in seminiferous tubules diameter (STsD),
while interstitial tissue thickness (ITT)
increased compared to that of control. Co-
administration of RJ significantly attenuated
the OXM-induced morphometric changes.
Data exist in Figure 2 revealed that
infiltration of mononuclear immune cells
(MNICs) in testicular tissue was significantly
elevated by OXM treatment. However, this
elevation in the number of MNICs was
inhibited by concurrent treatment with RJ.
Moreover, OXM treatment induced the
deletion of germ cells during
spermatogenesis, which resulted in significant
decreases in TDI and SCI (Table 3). Due to
the germ cells deletion, SPI and MI were also
greatly decreased in the OXM-treated mice
(Table 3). RJ treatment significantly
prevented the OXM-induced germ cell loss
from seminiferous tubules (Table 3).
Histopathologic findings
There were no marked histological
alterations in testes of control (Figure 3a) and
RJ-only (Figure 3b) groups, while drastic
morphologic changes were observed in the
testis of OXM-treated mice (Figure 3c). The
seminiferous tubules of these animals showed
severe hypocellularity and intraepithelial
vacuolization and were displaced by some
fibrinoid debris. Rupture, vacuolization,
inflammatory cells infiltration, and interstitial
space widening were also observed in
intertubular connective tissue of testes
following OXM treatment (Figure 3c). OXM-
induced lesions in testicular tissue were
greatly recovered by RJ co-administration,
although partial disorganizations were
observed in some seminiferous tubules
epithelium (Figure 3d).
Table 2. Effect of oxymetholone and Royal jelly on histological parameters of testis.
Control OXM RJ OXM+RJ
STsD (µm) 204.75±6.55 149.36±6.07
a
211.75±3.13
b
191.00±3.66
b
ITT (µm)
48.50±5.02 155.83±9.27
a
51.08±5.71
b
45.75±2.45
b
STsD, seminiferous tubules diameter; ITT, interstitial tissue thickness.
The values are expressed as mean ± S.E.M. (n = 8).
a Significant differences as compared with the control group at P <0.05
b Significant differences as compared with the OXM group at P <0.05
0
1
2
3
4
5
Control OXM Royal Jelly OXM+Royal Jelly
Testostrone(ng/ml)
a
ba,b

Protective Role of Royal Jelly in Oxymetholone… Iranian Journal of Toxicology
1077
http://www.ijt.ir; Volume 8, No 25, Summer 2014
Table 3. Effect of oxymetholone and Royal jelly on spermatogenic activities.
Control
OXM
RJ
OXM+RJ
TDI (%) 65.83±4.40 41.66±2.20
a
70.33±1.45
b
87.50±3.01
a,b
SPI (%) 70.88±2.73 41.83±1.73
a
68.88±2.93
b
69.99±1.92
b
SCI (%) 63.73±0.66 47.16±3.10
a
62.73±1.44
b
56.30±1.80
b
MI (%) 68.61±8.28 38.88±1.46
a
75.55±9.82
b
77.77±8.01
b
TDI, tubule differentiation index; SPI, spermiation index; SCI, Sertoli cell index; MI, miotic index.
The values are expressed as mean ± S.E.M. (n = 8).
a Significant differences as compared with the control group at P <0.05
b Significant differences as compared with the OXM group at P <0.05
Figure 2. Effect of oxymetholone and Royal jelly on numbers of mononuclear immune cells
(MNICs) in testicular tissue.
The values are expressed as mean ± S.E.M. (n = 8).
a Significant differences as compared with the control group at P <0.05
b Significant differences as compared with the oxymetholone group at P <0.05
Figure 3. Cross-sections of testes in mice treated with oxymetholone and/or royal jelly.
Testes from control (a) and RJ-treated (b) mice show intact seminiferous tubules with active
spermatogenesis. However, a testis from an OXM-treated mouse (c) reveals germ cells deletion and impaired
spermatogenesis. In the testis of a mouse treated with OXM and RJ, the seminiferous epithelium is less
abnormal as compared with (c), although partial disorganizations appear in some seminiferous tubules
epithelium.
Hematoxylin and eosin (×600)
0
2
4
6
8
Control OXM Royal Jelly OXM+Royal Jelly
Number of MNICs/mm2
a
b
a,b

Iranian Journal of Toxicology Gholamreza Najafi et al
1078 Volume 8, No 25, Summer 2014; http://www.ijt.ir
DISCUSSION
Synthetic AASs do the same work as
androgenic hormones do. Evidence exists that
these compounds have profound effects on
male endocrinological and reproductive
systems [37]. It has been reported that AASs
induce oligozoospermia and azoospermia (18)
as well as sperm morphology impairment
[16]. Previous study on male rats has also
confirmed the potential of AASs for causing
spermatogenic arrest [14]. Although the
mechanism by which AASs cause
reproductive toxicities is still under debate,
impaired spermatogenesis is supposed to
occur because of AASs-induced
hypogonadotrophic hypogonadism [17, 18].
Spermatogenesis is a complex process,
where spermatogonia develop into highly
differentiated spermatozoa through several
strictly controlled steps [38] and histological
parameters, such as STsD and ITT along with
TDI, SPI, SCI and MI, can give information
about the testicular damage degree as a
consequence of germ cell death. In general,
massive germ cell loss caused by reproductive
toxicity is followed by considerable
alterations in testicular histological
parameters. As shown in the present study,
depletion of seminiferous epithelium and the
consequent changes in histological
measurements caused by OXM were
confirmed in our report.
In the present study, OXM treatment
caused a marked reduction in serum
testosterone level, confirming a previous
report that AASs, especially 17α-alkylated
steroids, induced marked depressions of
serum testosterone and sex hormone-binding
globulin by means of androgenic receptors
occupation [39].
Sertoli cells (SCs) are the main somatic
cells of testis which play a major role in
cytoarchitectural organization of the
seminiferous tubules and, more importantly,
govern the differentiation of germ cells
(GCs). The physical and functional supports
of SCs are essential for GCs survival and
development [1, 2]. Therefore, a potential
explanation for the failure of spermiogenesis
in the OXM-treated mice is disruption of
testosterone-dependent junction of SCs with
GCs leading to their disorganization and
separation.
MNICs infiltration in testicular tissue is
an indicator of inflammation, a complex
clinical condition that can negatively affect
reproductive potential, which shows some
alterations occur at the suspected testis.
Hence, the elevation in MNICs infiltration
observed in testicular tissue of OXM-treated
animals might reflect the role of OXM in
induction of inflammation.
To date, several reports have supported
the fact that RJ has a predominant repro-
protective role in humans and animals.
Recently, it has been revealed that oral
administration of RJ counters “summer
infertility” in male rabbits [24]. Furthermore,
results of a study on 99 couples with
asthenospermia-induced infertility have
revealed that a simple and efficient way of
treating this condition is the intravaginal
administration of RJ and honey [25].
Additionally, it has been found that RJ has a
potential positive effect on development of
genital organ in male mice due to its highly
efficient anti-oxidant properties [27].
In the present study, RJ co-
administration provided effective protection
against OXM-induced oxidative injuries in
mouse testis. This reinforces the fact that RJ
as a potent and safe antioxidant has beneficial
effects against oxidative stress–related
toxicities. The reason why RJ co-
administration attenuated OXM-induced
reproductive toxicity could be attributed to
the fact that it contains spermatogenesis-
stimulating substances such as vitamin C,
vitamin E, and arginine [40]. Moreover, it has
been demonstrated that RJ inhibits the
production of pro-inflammatory cytokines by
activated macrophages [41].
CONCLUSION
In sum, it can be concluded that RJ has
a protective effect against OXM-induced
reproductive toxicities through restoration of
antioxidant defense system.
ACKNOWLEDGMENT
The authors gratefully acknowledge the
financial assistance of Urmia University in
performing this investigation.

Protective Role of Royal Jelly in Oxymetholone… Iranian Journal of Toxicology
1079
http://www.ijt.ir; Volume 8, No 25, Summer 2014
REFERENCES
1. Matzuk MM, Lamb DJ. The biology of
infertility: research advances and clinical
challenges. Nature medicine.
2008;14(11):1197-213.
2. Ikawa M, Tergaonkar V, Ogura A, Ogonuki
N, Inoue K, Verma IM. Restoration of
spermatogenesis by lentiviral gene transfer:
offspring from infertile mice. Proceedings of
the National Academy of Sciences.
2002;99(11):7524-9.
3. Schaison G, Young J, Pholsena M, Nahoul K,
Couzinet B. Failure of combined follicle-
stimulating hormone-testosterone
administration to initiate and/or maintain
spermatogenesis in men with
hypogonadotropic hypogonadism. The
Journal of clinical endocrinology and
metabolism. 1993;77(6):1545-9.
4. Hsieh M-L, Huang S-T, Huang H-C, Chen Y,
Hsu Y-C. The reliability of ultrasonographic
measurements for testicular volume
assessment: comparison of three common
formulas with true testicular volume. Asian
journal of andrology. 2009;11(2).
5. Jiang Y-g, Peng T, Luo Y, Li M-c, Lin Y-h.
Resveratrol reestablishes spermatogenesis
after testicular injury in rats caused by 2, 5-
hexanedione. Chinese Medical Journal
(English Edition). 2008;121(13):1204-9.
6. Prakash S, Prithiviraj E, Suresh S.
Developmental changes of seminiferous
tubule in prenatal, postnatal and adult testis of
bonnet monkey (Macaca radiata). Anatomia,
histologia, embryologia. 2008;37(1):19-23.
7. Sakamoto H, Yajima T, Nagata M, Okumura
T, Suzuki K, Ogawa Y. Relationship between
testicular size by ultrasonography and
testicular function: measurement of testicular
length, width, and depth in patients with
infertility. International journal of urology.
2008;15(6):529-33.
8. Yesalis CE, Kennedy NJ, Kopstein AN,
Bahrke MS. Anabolic-androgenic steroid use
in the United States. Jama.
1993;270(10):1217-21.
9. Pavlatos AM, Fultz O, Monberg MJ, Vootkur
A. Review of oxymetholone: a 17α-alkylated
anabolic-androgenic steroid. Clinical
therapeutics. 2001;23(6):789-801.
10. Hosseinimehr SJ, Zakaryaee V, Froughizadeh
M. Oral oxymetholone reduces mortality
induced by gamma irradiation in mice through
stimulation of hematopoietic cells. Molecular
and cellular biochemistry. 2006;287(1-2):193-
9.
11. Hengge UR, Stocks K, Wiehler H, Faulkner
S, Esser S, Lorenz C, et al. Double-blind,
randomized, placebo-controlled phase III trial
of oxymetholone for the treatment of HIV
wasting. AIDS. 2003;17(5):699-710.
12. Karila T, Karjalainen J, Mäntysaari M,
Viitasalo M, Seppälä T. Anabolic androgenic
steroids produce dose-dependent increase in
left ventricular mass in power athletes, and
this effect is potentiated by concomitant use
of growth hormone. International journal of
sports medicine. 2003;24(05):337-43.
13. Mooradian AD, Morley JE, Korenman SG.
Biological actions of androgens. Endocrine
Reviews. 1987;8(1):1-28.
14. Kind FA, Maqueo M, Dorfman RI. Influence
of various steroids on testes and accessory sex
organs in the rat. Acta endocrinologica.
1965;49(1):145-54.
15. Klaiber EL, Henzl MR, Lloyd CW, Segre EJ.
Corpus Luteum-Inhibiting Action of
Oxymetholone. The Journal of Clinical
Endocrinology & Metabolism.
1973;36(1):142-7.
16. Torres-Calleja J, Gonzalez-Unzaga M,
DeCelis-Carrillo R, Calzada-Sanchez L,
Pedron N. Effect of androgenic anabolic
steroids on sperm quality and serum hormone
levels in adult male bodybuilders. Life
sciences. 2001;68(15):1769-74.
17. Jarow JP, Lipshultz LI. Anabolic steroid-
induced hypogonadotropic hypogonadism.
The American journal of sports medicine.
1990;18(4):429-31.
18. Schürmeyer T, Belkien L, Knuth U,
Nieschlag E. Reversible azoospermia induced
by the anabolic steroid 19-nortestosterone.
The lancet. 1984;323(8374):417-20.
19. Karaali A, Meydanoglu F, Eke D. Studies on
composition, freeze-drying and storage of
Turkish royal jelly. Journal of apicultural
research. 1988; 27: 182-5.
20. Guo H, Ekusa A, Iwai K, Yonekura M,
Takahata Y, Morimatsu F. Royal jelly
peptides inhibit lipid peroxidation in vitro and
in vivo. Journal of nutritional science and
vitaminology. 2008;54(3):191-5.
21. Hattori N, Nomoto H, Fukumitsu H, Mishima
S, Furukawa S. Royal jelly and its unique
fatty acid, 10-hydroxy-trans-2-decenoic acid,
promote neurogenesis by neural
stem/progenitor cells in vitro. Biomedical
research (Tokyo, Japan). 2007;28(5):261-6.
22. Fujiwara S, Imai J, Fujiwara M, Yaeshima T,
Kawashima T, Kobayashi K. A potent
antibacterial protein in royal jelly. Purification
and determination of the primary structure of

Iranian Journal of Toxicology Gholamreza Najafi et al
1080 Volume 8, No 25, Summer 2014; http://www.ijt.ir
royalisin. Journal of biological chemistry.
1990;265(19):11333-7.
23. Šver L, Oršolić N, Tadić Z, Njari B, Valpotic
I, Bašic I. A royal jelly as a new potential
immunomodulator in rats and mice.
Comparative immunology, microbiology and
infectious diseases. 1996;19(1):31-8.
24. Elnagar SA. Royal jelly counteracts
bucks’“summer infertility”. Animal
reproduction science. 2010;121(1):174-80.
25. Abdelhafiz AT, Muhamad JA. Midcycle
pericoital intravaginal bee honey and royal
jelly for male factor infertility. International
Journal of Gynecology & Obstetrics.
2008;101(2):146-9.
26. Husein M, Haddad S. A new approach to
enhance reproductive performance in sheep
using royal jelly in comparison with equine
chorionic gonadotropin. Animal reproduction
science. 2006;93(1):24-33.
27. Kato A, Onodera M, Ishijima Y. Effect of
royal jelly on development of genital organ in
male mice. Journal of the Tokyo Society of
Veterinary and Zootechnical Science (Japan).
1988; 35: 1-4.
28. Aebi H. Catalase in vitro. Methods Enzymol.
1984;105:121-6.
29. Esterbauer H, Cheeseman KH. Determination
of aldehydic lipid peroxidation products:
malonaldehyde and 4-hydroxynonenal.
Methods in enzymology. 1990;186:407-21.
30. Qin D, Lung MA. Morph metric study on
Leydig cells in capsuletomized testis of rats.
Asian J Androl. 2002; 4: 49-53.
31. Vendramini V, Sasso-Cerri E, Miraglia SM.
Amifostine reduces the seminiferous
epithelium damage in doxorubicin-treated
prepubertal rats without improving the
fertility status. Reproductive Biology and
Endocrinology. 2010;8(1):3.
32. Gundersen HJG. Notes on the estimation of
the numerical density of arbitrary profiles: the
edge effect. Journal of microscopy.
1977;111(2):219-23.
33. Porter KL, Shetty G, Meistrich ML.
Testicular edema is associated with
spermatogonial arrest in irradiated rats.
Endocrinology. 2006;147(3):1297-305.
34. Rezvanfar M, Sadrkhanlou R, Ahmadi A,
Shojaei-Sadee H, Mohammadirad A, Salehnia
A, et al. Protection of cyclophosphamide-
induced toxicity in reproductive tract
histology, sperm characteristics, and DNA
damage by an herbal source; evidence for role
of free-radical toxic stress. Human &
experimental toxicology. 2008;27(12):901-10.
35. Kang J-K, Lee Y-J, No K-O, Jung E-Y, Sung
J-H, Kim Y-B, et al. Ginseng intestinal
metabolite-I (GIM-I) reduces doxorubicin
toxicity in the mouse testis. Reproductive
Toxicology. 2002;16(3):291-8.
36. Kheradmand A, Dezfoulian O, Tarrahi MJ.
Ghrelin attenuates heat-induced degenerative
effects in the rat testis. Regulatory peptides.
2011;167(1):97-104.
37. Ruokonen A, Alen M, Bolton N, Vihko R.
Response of serum testosterone and its
precursor steroids, SHBG and CBG to
anabolic steroid and testosterone self-
administration in man. Journal of steroid
biochemistry. 1985;23(1):33-8.
38. Tanaka SS, Toyooka Y, Akasu R, Katoh-
Fukui Y, Nakahara Y, Suzuki R, et al. The
mouse homolog of Drosophila Vasa is
required for the development of male germ
cells. Genes & development. 2000;14(7):841-
53.
39. Sader MA, Griffiths KA, McCredie RJ,
Handelsman DJ, Celermajer DS. Androgenic
anabolic steroids and arterial structure and
function in male bodybuilders. Journal of the
American College of Cardiology.
2001;37(1):224-30.
40. Hassan A. Effect of royal jelly on sexual
efficiency in adult male rats. Iraqi Journal of
Veterinary Sciences. 2009;23: 155-60.
41. Kohno K, Okamoto I, Sano O, Arai N, Iwaki
K, Ikeda M, et al. Royal jelly inhibits the
production of proinflammatory cytokines by
activated macrophages. Bioscience,
biotechnology, and biochemistry.
2004;68(1):138-45.