Int. J. Biosci.

2015
International Journal of Biosciences | IJB |
ISSN: 2220-6655 (Print), 2222-5234 (Online)
http://www.innspub.net
Vol. 6, No. 5, p. 70-78, 2015

RESEARCH PAPER

OPEN ACCESS

Busulfan induces apoptotic and cytotoxic effects on testis and

epididymal sperm of adult male mouse following low dose
treatment
Akbar Vahdati1,2, Ali Reza Fathi1,2, Parva Nasimi1,2*, Ghasem Saki3
1

Department of Biology, Fars Science and Research Branch, Islamic Azad University, Fars, Iran

Department of Biology, Shiraz Branch, Islamic Azad University, Shiraz, Iran

Department of Anatomy, School of Medicine, Jondishapour University of Medical Sciences, Ahwaz,

Iran
Key words: Apoptosis, Busulfan, Epididymal Sperm, Seminiferous tubule, Sperm viability.

http://dx.doi.org/10.12692/ijb/6.5.70-78

Article published on March 09, 2015

Abstract
At adequate concentrations busulfan selectively attacks the dividing spermatogonia and spermatocytes. But,
there is not enough information about side effects of low dose busulfan. The aim of this study was to assess
changes that occur on seminiferous tubules morphology and epididymal sperm of adult male mouse following
treatment with low dose busulfan. So, adult male NMRI mice (25-35 g) were assigned in three groups including;
Group 1: Control, Group 2; treated with busulfan for 21 days (0.06 mg/kg/day) and Group 3: treated with
busulfan for 21 days (0.8 mg/kg/day). The effects of busulfan on seminiferous tubules morphology and
epididymal sperm were evaluated by light microscopy, ComputerAided Sperm Analysis (CASA), MTT assay and
flowcytometry. The results showed that busulfan caused significant germinal epithelium destruction in treated
mice versus control. Also, busulfan had significant cytotoxic and apoptotic effects in epididymal sperm of treated
mice. The percentages of early apoptotic, late apoptotic and necrotic sperms in groups 2 and 3 demonstrated
significant increase versus control. Also, analysis of sperm parameters and sperm viability using CASA and MTT
assay demonstrated significant differences between control and busulfan treated mice. In conclusion,
chemotherapy with low dose busulfan causes significant changes on sperm viability, sperm parameters and
morphology of seminiferous tubules. All side effects of busulfan are dose dependent and they are considerable in
mice treated with 0.8 mg/kg busulfan. Hence, the treatment with low dose busulfan may reduce toxicity of
chemotherapy.
* Corresponding

Author: Parva Nasimi Parvanasimi62@yahoo.com

70 Vahdati et al.

Int. J. Biosci.

2015

Introduction

In addition, the study of underlying mechanisms that

Busulfan [CH3SO2O (CH2)4OSO2 CH3] has been

anticancer agents induced cytotoxicity is necessary for

widely used for the treatment of patients with chronic

reducing side effect of chemotherapy. Hence, our

myelogenous leukemia and prior to bone marrow

present study is to evaluate busulfan mediated

transplantation (Down and Ploemacher, 1993; Buggia

damage on epididymal sperm and testis tissue of

et al., 1994). The treatment plan for busulfan depends

adult male NMRI mice following low dose treatment.

on the type of cancer (Perry, 2012).

Methods and materials
It seems that busulfan has various long term or late

Experimental animals

effects and its genotoxicity may be one reason

Adult NMRI male mice (n = 30) were included in the

accounting for these effects (Blasiak et al., 1999;

present study. The mice were 8 weeks old and

Blasiak et al., 2000). In addition, recent studies

weighed 25-35 g each. They were purchased from

showed

Ahwaz Medical Sciences Research

long-term

effects

of

busulfan

on

spermatogenesis (Temitope et al., 2011).

Center

and

Experimental Animal House (Ahwaz, Jondishapour

University of Medical Sciences, Iran) and were

This chemotherapeutic agent affects different body

allowed to adapt themselves to the new conditions for

organs such as gonads and also increases percentage

one week.

of germ cell apoptosis of various mammalian (Choi et

al., 2004; Vaisheva et al., 2007; Mohammad-

The animals were housed in temperature controlled

Ghasemi et al., 2009). Male germ cell apoptosis is a

rooms (25C) with constant humidity (40-70%) and

persistent effect of busulfan that reported extensively

12 h light/ dark cycle prior to experimental protocols.

in mouse and murine (Mohammad-Ghasemi et al.,

They were provided rat chow (Pars, Tehran, Iran) and

2009).

water at libitum. All animals used were cared for

according to the Guide for the Care and Use of

Busulfan acts as an alkylating agent and primarily

Laboratory Animals by the National Academy of

targets slowly proliferating or non-proliferating cells

Sciences (National Institutes of Health publication

(Wenzhi et al., 2011). Also, busulfan induces DNA

No. 86-23).

alkylation leading to DNA-DNA and DNA-protein

cross-links in sperm (Iwamoto et al., 2004; Mertins et

Treatment protocol

al., 2004).

Busulfan (Sigma, St. Louis, MO) was dissolved into

DMSO (<0.2 %) (Sigma, St. Louis, MO) and diluted

Mohammad-Ghasemi et al (2009) reported that

with sterile deionized water (1 : 1) at room

busulfan administration in a different single dose of

temperature to provide final concentrations of 0.06

10, 20 and 40 mg/kg induces side effect on male

and 0.8 mg/kg. Dosage of busulfan was chosen based

reproductive system (Mohammad-Ghasemi et al.,

on previous studies (Fernandez et al., 2002; Bruce et

2009).

al., 2011). Busulfan was daily prepared and freshly

used during the experiment.

Also, there are many reports about side effects of

chemotherapic agents on normal tissue. But, they

Male adult NMRI mice were divided into three groups

studied side effects of high or lethal doses of these

of ten each. Group 1 (control) was administered

agents on reproductive system (Choi et al., 2004;

DMSO for 21 days, groups 2 and 3 were given

Mohammad-Ghasemi et al., 2009). In the other hand,

respectively 0.06 and 0.8 mg/kg busulfan for 21 days

there is no strong study which determined various

intraperitoneally. At the end of the experiment,

data about effects of chemotherapic agents on

animals were anesthetized using 80 mg/kg ketamine

spermatogenesis.

hydrochloride and 10 mg/kg xylazine, killed by

71 Vahdati et al.

Int. J. Biosci.

2015

decapitation and the left testis and epididymis were

Dehydrogenase in mitochondria converts yellow

removed.

colored insoluble tetrazolium salt to purple colored

water-soluble

formazan.

Sperm

also

have

Collection of sperm

mitochondria in the midpiece; therefore sperm

Cauda-epididymis was dissected; several longitudinal

viability could be evaluated by MTT reduction assay

incisions were made on its distal end and placed into

(Byum et al., 2008).

a 60 mm tissue culture dish containing warmed 1 ml

PBS (pH 7.4). Then the tissue was removed and the

Recent studies showed that HAMS F10+25mM

sperm suspension was used for ComputerAided

HEPES (Gibco) is the most suitable media for the

Sperm Analyzer, MTT assay and flowcytometry.

sperm MTT viability assay (Nasr-Esfahani et al.,

2002). Thus routinely MTT (Loba Chemie, India) was

Histopathology and Light microscopy

dissolved in HAMSF10+25mMHEPES at 0.5 mg/mL

The left testes were fixed in 10% formalin and

and then pH was adjusted to 7.47.45. BSA was

embedded in paraffin. Five-micron thick sections

added to MTT solution at 10% concentration. MTT

were prepared and stained with Hematoxylin and

solution was kept at 4C in the dark for a maximum of

Eosin (H&E). The specimens were examined under

1 week. For routine MTT assay 50 l of washed sperm

Olympus/3H light microscope-Japan.

(1.5-2.5 millions) was added to 450 l of MTT

solution, which was warmed to 37C, 30 min before

This analysis examined morphologically whether

addition of sperm.

busulfan induced cell depletion and destruction on

seminiferous tubule of mice that treated with low

The absorbance was measured with a microtiter plate

dose busulfan.

reader (Bio-Tek SX2, Winooski, VT, USA) at a test

wavelength of 570 nm and a reference wavelength of

The severity of seminiferous tubule destruction was

690 nm. The optical density (OD) was calculated as

determined according to two factors; germinal

the difference between the absorbance at the

epithelium depletion and existence of sperm in

reference wavelength and that at the test wavelength.

seminiferous tubule. So, we determined four types of

Results were expressed as percentage of control.

seminiferous tubules; type 1) normal germinal

epithelium with sperm, type 2) germinal epithelium

FITC Annexin V/PI Assay; flowcytometry

depletion

Flowcytometry

and

with

sperm,

type

3)

germinal

measures

apoptosis

and

epithelium depletion and without sperm, type 4)

phosphatidylserine (PS) translocation across the

empty seminiferous tubule. The tubular diameter of

plasma membranes (Anzar et al., 2002; Hossain et

the seminiferous tubule epithelium was using image

al., 2011).

analyzer Leica (DMLB) and Leica Qwin software.

FITC Annexin V/Dead Cell Apoptosis Kit (Catalog no.
Sperm parameters

V13242,

Invitrogen)

was

used

to

detect

the

Sperm parameters including sperm count and sperm

translocation of PS and type of cell death on

abnormality were monitored by CASA. Also, sperm

epididymal sperm of control and busulfan-treated

parameters were compared in both control and

mice.

busulfan treated groups.

Sperm sample was centrifuged (500 g, 10 min and
Assessment of Cell Viability: MTT assay

25C). The pellet was resuspended in 1X Annexin-

MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl

binding buffer. 5 L of FITC Annexin V (Component

tetrazolium bromide) reduction assay is a method

A) and 1 L of the 100 g/mL PI working solution

that validates the

were added to each 100 L of cell suspension and

viability of an active cell.

72 Vahdati et al.

Int. J. Biosci.

2015

incubated at room temperature for 15 minutes. After

were expressed as mean SD. The statistical

the incubation period, 400 L of 1X Annexin-binding

significances were analyzed by analysis of variance

buffer was added to tube, mixed gently and the

(ANOVA) and Tukey post-hoc analyses. In all cases,

sample kept on ice. As soon as possible, the stained

P<0.05 was considered significant.

cells were analyzed by flow cytometry and the

fluorescence emission was measured at 530 nm and >

Results

575 nm. Flowcytometric evaluation was conducted

Seminiferous tubule morphology

within 5 min.

Morphological appearance of seminiferous tubule was

compared between control and treated groups using

Statistical analysis

light microscope (Fig. 1).

Data analyses were done using the SPSS 16.0 software

package (SPSS Inc., Chicago, IL, USA). The results
Table 1. The results of sperm count, normal morphology and sperm pathology of control and busulfan treated
groups.
Groups

Sperm Count106

Normal Morphology%

Head Pathology%

Neck pathology% Tail Pathology%

Control

10.13 1.2 a

81.7 6.9 a

11.1

12.3

5.3

Group 2

7.3 0.61

68.9 7.1

22,7

13,6

16.1

Group 3

2.5 0.4

21.5 3.4

50.5

20.3

14.2

Different letters indicate significant differences between groups based one-way ANOVA (P < 0.05).
Microscopic observations of seminiferous tubule

68.2 7.3%, respectively (Fig. 2A). In addition,

demonstrated

diameter

significant

changes

on

germinal

of

seminiferous

tubule

decreased

epithelium of busulfan treated mice (groups 2 and 3).

remarkably in treated groups with low dose busulfan.

In these groups, germinal epithelium showed high

Diameter of seminiferous tubules in control, groups 2

level of destruction. Percentage of type 3 seminiferous

and 3 were 838.5 43.9, 795.4 31.4 and 756.7

tubule (with destroyed germinal epithelium and no

29.6 m, respectively (fig. 2B). But, all seminiferous

sperm) in groups 2 and 3 were about 21.9 5.8% and

tubules in control (group 1) were normal (type 1).

Table 2. The percentages of early apoptotic, late apoptotic and necrotic sperm in control and busulfan treated
mice.
Groups

Early Apoptotic Sperms %

Late Apoptotic Sperms %

Necrotic Sperms %

Control

1.8 0.5 a

1.2 0.3 a

0.3 0.1 a

Group 2

11.8 0.9 b

8.2 1.5 b

2.1 0.5 b

Group 3

27.6 2 c

19.1 3.1 c

3.2 0.9 b

Different letters indicate significant differences between groups based one-way ANOVA (P < 0.05).
Sperm parameters

Also, sperm pathology increased in treated group

The results showed remarkable differences on sperm

compared

count, normal morphology and sperm pathology

demonstrated highest percentage of head and neck

(abnormality) between control and treated groups

pathologies. But, group 2 had more tail pathology

(Tab. 1). Percentages of sperm count (Fig. 3) and

between 3 groups. In addition, teratospermia was

morphologically normal sperm (Fig. 4A) were lower

remarkable in treated groups in comparison with

in busulfan treated groups versus control.

control (Fig. 4B).

73 Vahdati et al.

with

control

(Tab.

1).

Group

Int. J. Biosci.

2015

Assessment of Cell Viability: MTT assay

groups in comparison with untreated mice (Fig. 5).

MTT assay was used to examine the cytotoxicity of

Percentages of sperm viability were 72.4 9.6% and

busulfan on epididymal sperm viability of control and

57.5 5.2% in groups 2 and 3, respectively. Sperm

treated mice. Busulfan treated groups showed high

viability decreased in treated groups notably. Also,

level of sperm cell death. Our results determined

this reduction was dose dependent.

significant differences on sperm viability of treated

Fig. 1. Light microscopy images of seminiferous tubule in control and busulfan treated mice. A) Seminiferous
tubules with normal germinal epithelium. B) Slight decrease of germ cell population and normal lumen in group
2. C) Atrophy of seminiferous tubule with widening of lumen, depletion of germinal epithelium and massive germ
cell loss in group 3 (Arrows are showing germinal epithelium depletion).
FITC Annexin V/PI Assay; flowcytometry

The

results

illustrated

remarkable

differences

Flowcytometry separated the population of sperms

between busulfan treated mice and control (Tab. 2).

into four groups: live cells showed only a low level of

The percentages of early apoptotic, late apoptotic and

fluorescence, necrotic cells showed red fluorescence,

necrotic sperms in groups 2 and 3 increased

early apoptotic cells showed green fluorescence and

significantly versus control (Fig. 7).

late apoptotic cells showed both red and green

fluorescence (Fig. 6).

Discussion
Recent studies demonstrated that chemotherapy

Sperms were incubated with FITC Annexin V in a

drugs such as busulfan can cause testicular damage as

buffer which contained propidium iodide (PI) and

manifested

analyzed by flow cytometry. Untreated cells were

oligozoospermia and apoptotic cell death on testicular

primarily FITC Annexin V and PI negative, indicating

germinal epithelium (Mohammad-Ghasemi et al.,

that they were viable and not undergoing apoptosis.

2009). Busulfan unlike other chemicals primarily

There were two populations of cells (bottom panels):

destroys spermatogonial stem cells. But, other

Cells that were viable and not undergoing apoptosis

chemicals except of busulfan kill differentiated

or necrosis (FITC Annexin V and PI negative) and

spermatogonia (Kanatsu-Shinohara et al., 2003;

cells undergoing apoptosis (FITC Annexin V positive

Anjamrooz et al., 2007; Kawashima et al., 2009). A

and PI negative). The population of sperms was

single intraperitoneal injection of 10, 20, 30, 40 and

observed to be FITC Annexin V and PI positive,

50 mg/kg of busulfan revealed the deletion of the

indicating that they were in end stage apoptosis or

spermatogenic

already dead. Also, a population of sperms was shown

infertility in several species of animals (Anjamrooz et

to be FITC Annexin V negative and PI positive, they

al., 2007; Kawashima et al., 2009). Also, the duration

underwent necrosis (Fig. 6).

of busulfan induced infertility is dependent on the

74 Vahdati et al.

by

reduced

cells

and

testicular

induces

volume,

permanently

Int. J. Biosci.

2015

extent of stem cell depletion (Kanatsu-Shinohara et

days) had lower sperm viability versus control and

al., 2003). Agarwa et al (2003) suggested that high

treated mice with 0.06 mg/kg busulfan (for 21 days).

level of sperm DNA damage can be seen following a

Furthermore, apoptotic and necrotic cell deaths

single dose of chemotherapeutic drugs, which may

increased on epididymal sperms of busulfan treated

persist for several months after cessation of their use

groups compared with control (p< 0.05). The level of

(Agarwa et al., 2003; Mohammad-Ghasemi et al.,

apoptotic sperm was interestingly in treated groups

2009).

(groups 2 and 3), but, the level of necrotic sperm was

not notable in these groups. The extent of apoptotic
sperm in group 3 may be related to dose-dependent
effects of busulfan.

Fig. 3. Analysis of sperm count (106/ml) in control

and busulfan treated mice. There were significant
decline on sperm count of groups 2 (0.06 mg/kg/day
busulfan for 21 days) and 3 (0.8 mg/kg/day busulfan
for 21 days) in comparison with control. Different
letters indicate significant differences between group
Fig. 2. Analysis of germinal epithelium depletion and

based one-way ANOVA (P< 0.05). Bu: Busulfan.

seminiferous tubule diameter in control and busulfan

treated mice. A) Percentages of germinal epithelium

percentages of morphologically normal sperm in

depletion in treated groups showed significant

treated groups was notable. Although there was no

changes in comparison with control, B) There was no

remarkable change between control and group 2, but,

significant change on seminiferous tubule diameter of

group 3 showed significant changes compared with

group 2 in comparison with control. But, group 3

control. B) There were remarkable differences in the

revealed significant decrease versus control. Different

percentage of sperm pathology between control and

letters indicate significant differences between groups

busulfan treated mice. Group 3 showed the highest

at P< 0.05.

level of sperm pathology and the lowest level of

normal morphology versus control and group 2.

Whereas previous researches which were shown

Different

destructive effects of high single dose of busulfan on

between groups based one-way ANOVA (P< 0.05).

letters

indicate

significant

differences

testicular germinal epithelium structure, present

Bu: Busulfan.

study revealed busulfan-mediated side effects on

seminiferous tubule morphology following low dose

In the other hand, our flowcytometric results strongly

chemotherapy.

emphasized high level of DNA damage on epididymal

sperm of treated mice with low dose busulfan. DNA

In addition, MTT assay demonstrated lower sperm

fragmentation as an important feature of apoptosis

viability in treated groups in comparison with control.

(Nasimi and Roohi, 2012) was remarkable in treated

The mice that received 0.8 mg/kg busulfan (for 21

groups with low dose busulfan.

75 Vahdati et al.

Int. J. Biosci.

2015
dose

busulfan

were

due

to

cytotoxicity

of

chemotherapy on spermatogenesis and confirmed the

results of flowcytometry and MTT assay.

Fig. 5. MTT assay evaluated sperm viability in

control and busulfan treated mice. Sperm viability
Fig. 4. The percentages of morphologically normal

decreased significantly in groups 2 and 3 versus

sperm and sperm pathology in control and busulfan

control.

treated mice. A) Difference in the Also, the high levels

differences between groups based one-way ANOVA

of sperm abnormality following treatment with low

(P< 0.05). Bu: busulfan.

Different

letters

indicate

significant

Fig. 6. The flowcytometric profile of epididymal sperm in control and busulfan treated mice that labeled with
Annexin V and PI. A) Necrotic cells labeled with PI but not with Annexin V-FITC. B) Late apoptotic cells with
significant green and red fluorescence. C) Nonfluorescent, fully viable cells. D) Early Apoptotic cells labeled with
Annexin V-FITC but not with PI.
necrotic sperm demonstrated significant changes
between control and busulfan treated mice. Different
letters indicate significant differences between groups
based one-way ANOVA (P < 0.05). Bu: Busulfan.
The results of sperm parameters, MTT assay and
flowcytometry revealed cytotoxic, genotoxic and
apoptotic effects of low dose busulfan on epididymal
sperm
Fig. 7. The flowcytometric results of epididymal
sperm in control and busulfan treated mice. The
percentages of early apoptotic, late apoptotic and

76 Vahdati et al.

of

busulfan

treated

mice.

But,

these

cytotoxicity and genotoxicity were lower than high

dose busulfan.

Int. J. Biosci.

2015

So, there are clear reasons that show chemotherapy

Practice. Fifth edition. Lippincott Williams & Wilkins

harms most organs of body, but our observations

press. Section 1.

illustrate that treatment with low dose busulfan has

lower cytotoxicity and genotoxicity in normal cell.

Buggia I, Locatelli F, Regazzi MB, Zecca M.

1994. Busulfan. Annual Pharmacotherapy 28, 1055-

Acknowledgments

1062.

This work was supported by the grant from Fars

Science

and

Research

Branch,

Islamic

http://dx.doi.org/10.1177/106002809402800911

Azad

University, Fars, Iran.

Byum Wj, Choo HS, Kim HH, Kim YJ, Hwang

YJ, Kim DY. 2008. Evaluation of Boar Sperm

References

Viability by MTT Reduction Assay in Beltsville

Agarwa A, Tamer MS, Said TM. 2003. Role of

Thawing Solution Extender. Asian-Australian journal

sperm chromatin abnormalities and DNA damage in

of animal science 21, 494-498.

male infertility. Oxford reviews of reproductive

http://dx.doi.org/10.5713/ajas.2008.70480

biology 9, 331-34.
http://dx.doi.org/10.1093/humupd/dmg027

Choi YJ, Ok DW, Kwon DN, Chung JI, Kim

HC, Yeo SM, Kim T, Seo HG, Kim JH. 2004.

Anjamrooz SH, Movahedin M, Javad Mowla S,

Murine male germ cell apoptosis induced by busulfan

Bairanvand

of

treatment correlates with loss of c-kit-expression in a

Morphological and Functional Changes in the Mouse

Fas/FasL- and p53-independent manner. FEBS

Testis and Epididymal Sperms Following Busulfan

Letters 575, 41-51.

Treatment. Iranian biomedical journal 11, 15-22.

http://dx.doi.org/10.1016/j.febslet.2004.08.034

Anzar E, He L, Buhr MM, Kroetsch TG, Pauls

Down JD, Ploemacher RE. 1993. Transient and

KP.

permanent

2002.

SP.

2007.

Sperm

Cryopreserved

Bull

Assessment

Apoptosis
Semen

in

Fresh

Detected

by

and
Flow

engraftment

potential

of

murine

hematopoietic stem cell subsets: differential effects of

Cytometry and Its Relationship with Fertility. Biology

host

of reproductive 66, 354-360.

cytotoxic drugs. Experimental Hematology 21, 913-

http://dx.doi.org/10.1095/biolreprod66.2.354

921.

Blasiak

J,

Kowalik

J,

Malecka-Panas

conditioning

with

gamma

radiation

and

E,

Fernandez HF, Tran HT, Albrecht F, Lennon

Drzewoski J. 2000. DNA damage and repair in

S, Caldera H, Goodman MS. 2002. Evaluation of

human lymphocytes exposed to three anticancer

safety

platinum drugs. Teratogenesis carcinogenesis and

intravenous busulfan in a twice-daily or daily

mutagenesis 20, 119-31.

schedule to patients with advanced hematologic

http://dx.doi.org/10.1002/(SICI)15206866(2000)20:

malignant

3<119

transplantation. Biology of blood and marrow

and

pharmacokinetics

disease

of

undergoing

administering

stem

cell

transplantation 8, 486-92.
Blasiak J, Kowalik J, Trzeciak A, Wojewodzka
M. 1999. Cytotoxicity and DNA damage and repair in

Hossain S, Johannisson A, Wallgren M, Nagy

human lymphocytes exposed to three anticancer

S, Siqueira AP, Rodriguez-Martinez H. 2011.

platinum drugs. Neoplasma 46, 61-63.

Flow cytometry for the assessment of animal sperm

integrity and functionality: state of the art. Asian

Chabner

BA.

Longo

DL.

2011.

Chemotherapy and Biotherapy: Principle and

77 Vahdati et al.

Cancer

journal of andrology 13, 406-419.

http://dx.doi.org/10.1038/aja.2011.15

Int. J. Biosci.

2015

Iwamoto T, Hiraku Y, Ikawa S, Mizutani H,

Nasr-Esfahani MH, Aboutorabi R, Esfandiari

Kojima M, Kawanishi S. 2004. DNA intrastrand

E, Mardani M. 2002. Sperm MTT Viability Assay: A

cross-link at the 5'-GA-3' sequence formed by

New Method for Evaluation of Human Sperm

busulfan and its role in the cytotoxic effect. Cancer

Viability. Journal of assistant reproductive and

Science 95, 454-458.

genetic 19, 477-482.

http://dx.doi.org/10.1111/j.13497006.2004.tb03231.x
Nasimi P, Roohi S. 2012. Cell death in animal and
Kanatsu-Shinohara M, Toyokuni S, Morimoto

plants; Apoptosis, Necrosis & Autophagy. Islamic

T, Matsui S, Honjo T, Shinohara TL. 2003.

Azad University of Masjed Soleyman press, 11-18.

Functional Assessment of Self-Renewal Activity of

Perry MC. 2012. Perrys The Chemotherapy Source

Male Germline Stem Cells Following Cytotoxic

Book. Lippincott Williams and Wilkins press.

Damage and Serial Transplantation. Biology of

reproduction 68, 1801-1807.

Temitope

http://dx.doi.org/10.1095/biolreprod.102.012575

Khaled TB, Effiong UA, Adetola AA. 2011.

AA,

Tolulope OA,

Funlola CT,

Protective Effect of Tahitian Noni Juice on the

Kawashima A, Osman BAH, Takashima M,

Reproductive Functions of Male Wistar Rats Traeted

Kikuchi A, Kohchi S, Satoh E, Tamba M,

with Cyclophosphamide. International journal of

Matsuda M, Okamura N. 2009. CABS1 is a Novel

particle therapy 10, 39-43.

Calcium-Binding Protein Specifically Expressed in

Elongate Spermatids of Mice. Biology of reproduction

Vaisheva F. Delbes G. Hales BF. Robaire B.

80, 1293-1304.

2007. Effects of the chemotherapeutic agents for non-

http://dx.doi.org/10.1095/biolreprod.108.073866

hodgkin lymphoma, cyclophosphamide, doxorubicin,

vincristine, and prednisone (CHOP), on the male rat

Mertins SD, Myers TG, Holbeck SL, Medina-

reproductive system and progeny outcome. Journal of

Perez W, Wang E, Kohlhagen G, pommier Y,

Andrology 28, 578-587.

Bates SE. 2004. In vitro evaluation of dimethane

http://dx.doi.org/10.2164/jandrol.106.002428

sulfonate analogues with potential alkylating activity

and selective renal cell carcinoma cytotoxicity.

Wenzhi M, Lei A. Wu Z, Wang X, Guo M, Miao

Molecular cancer therapeutics 3, 849-860.

K, Tian J. 2011. Efficient and Safe Recipient

Preparation

Mohammad-Ghasemi

F,

Bahadori

MH,

for

Transplantation

of

Mouse

Spermatogonial Stem Cells: Pretreating Testes with

Faghani M, Soleimani Rad J. 2009. Buserelin

Heat Shock1. Biological Reproduction 85, 670-677.

Inhibits Apoptosis in Male Germ Cells Induced by

http://dx.doi.org/10.1095/biolreprod.110.089623

Busulfan in Mouse Testis. Journal of Iranian

anatomical Sciences 7, 45-54.

78 Vahdati et al.