Journal of Medicine and Medical Science Vol. 2(4) pp. 777-782, April 2011
Available online @http://www.interesjournals.org/JMMS
Copyright © 2011 International Research Journals
Full Length Research Paper
L-arginine augments oxidative stress in cryptorchid
testes of adult Sprague-Dawley rats
Duru FIOε, Olalekan OOα, Azu OO*β and Okoko IIα
α
Department of Anatomy, College of Medicine, University of Lagos-Nigeria.
Department of Anatomy, Faculty of Health Sciences, Niger Delta University, Yenagoa.
β
Department of Anatomy, College of Health Sciences, University of Uyo, Uyo.
ε
Accepted 25 March, 2011
Experimental cryptorchidism has been associated with increased apoptotic degeneration of
spermatogenic cells resulting in increased free radical production. The aim of this study is to determine
the effect of L-arginine on semen parameters, histology and the antioxidant activities on induced
cryptorchid rat testis. Twenty male Sprague-Dawley rats weighing between 150-250 g were divided into
four groups (A- D) each with 5 rats per group. Animals in groups A and C were made unilaterally
cryptorchid by anchoring the upper pole of the testis to the anterior abdominal wall. While group A was
treated with 15 mg/100 mg body weight of L-arginine, group C had no treatment. Group B animals
received the same dose of L-arginine but not made cryptorchid. Group D animals served as control and
received distilled water. A significant decrease (p<0.05) in the sperm count was observed in groups A, B
and C when compared with the control. Sperm motility declined significantly in groups A and C.
Seminiferous tubular atrophy, depletion of spermatogenic cells as well as infiltration of interstitial
spaces was common in cryptorchid rats as well as those treated with l-arginine. Serum testosterone
levels were significantly increased in group B whereas group C recorded a decline. Luteinizing
hormone levels were significantly increased in groups B and C whereas follicle stimulating hormone
levels were decreased in group C when compared with the control. MDA levels were significantly
increased in groups A and C while SOD activity declined significantly in these groups. Catalase activity
decreased significantly in group C when compared with the control while glutathione reductase activity
reduced significantly in groups A and C when compared with the control. These results indicate that
administration of 15 mg/100 mg BW of L-arginine causes testicular oxidative stress and do not improve
the outcome of cryptorchidism in adult male Sprague-Dawley rats.
Keywords: Cryptorchidism, l-arginine, testis, histology, antioxidants, hormones.
INTRODUCTION
Cryptorchidism results from failure of the testis to
descend into the scrotum. It is a primary congenital
illness affecting up to 3% of boys (De Miguel et al., 2001)
and a major risk factor for male infertility. In most male
mammals the scrotal temperature is always lower than
that of the abdomen hence allowing for the maintenance
of optimal environment for testicular function.
Cryptorchidism has been shown to induce heat stress
(Yin et al., 1997) which predispose to free radical
formation that is responsible for the infertility in these
*Corresponding author Email amechi2@yahoo.com
patients. The surgical induction of cryptorchidism in
experimental animals has been shown to cause
disruption of spermatogenesis leading to infertility (Saalu
et al., 2007).
L-arginine is the biological precursor of nitric oxide
(NO), an endogenous gaseous messenger molecule
involved in a variety of endothelium-dependent
physiological functions (Wu and Meininger, 2000)
including its critical role in cardiovascular protection and
immune support (Sunita et al., 2000). It is now evident
that an increase in NO and a decrease in total antioxidant
capacity occur with cryptorchidism and contribute to
accelerated germ cell loss. Administration of nitric oxide
synthase inhibitor, N-omega-nitro-L-arginine methyl ester,
attenuated apoptosis and improved spermatogenesis in
778 J. Med. Med. Sci.
experimental models of cryptorchidism (DeFoor et al.,
2004).
L-arginine decreases oxidative stress by reduction of
the vascular superoxide anion production with
improvement
of
endothelial
function
in
hypercholesterolaemic subjects (Kawano et al., 2002). In
several conditions where oxygen metabolites are thought
to mediate endothelial and myocardial injury, l-arginine
has protective effects that might be due to its direct
chemical interaction with oxygen radicals (Lass et al.,
2002). Moreover, dietary l-arginine supplementation
attenuates the oxidative stress induced by burn injury
with a better macrophage response (Tsai et al., 2002).
These beneficial effects of l-arginine have been attributed
to its dependent formation of NO within the endothelial
lining (Brandes et al., 2000).
Men with unilateral undescended testis have been
shown to father significantly more children than those
with bilateral undescended testes (Cendron et al., 1989)
and studies have pointed to the fact that unilateral
cryptorchidism induces germ cell apoptosis in the
ipsilateral testis (Wang et al., 1998; Zini et al., 1999;
Watts et al., 2000); attention is now focused on the
possible intervention of l-arginine in the treatment of
persistent cryptorchidism since it is required for normal
spermatogenesis (Tanimura, 1967). The outcome would
provide a better prognosis in terms of testicular function
in adulthood (Hadziselmovic, 2001). This work is
therefore conceived to determine the effects of l-arginine
supplementation on a unilateral cryptorchid testis of
matured male Sprague-Dawley rats.
Experimental protocol
Groups A to C were the experimental groups; in group A
the right testis was made cryptorchid and 15 mg/100 g larginine administered for 8 weeks. Group B was treated
with 15 mg/100 g l-arginine for 8 weeks while in Group C
right testis made cryptorchid only with no treatment.
Group D served as the control and the rats were neither
rendered cryptorchid nor treated with l-arginine.
To induce right cryptorchidism, each rat was
anaesthetized and a small incision was made in the
upper abdomen. General anesthesia was given with 10
mg/kg of ketamine (Tsai et al., 2002). The right testis was
gently brought up into the abdominal cavity and fixed on
the upper abdominal wall using a 4-0 Nylon suture
passing through the connective tissue of the caput
epididymis as previously described in Jegou et al. (1984).
All the animals subsequently recovered fully. At the end
of the experimental period, all rats were sacrificed the
next day by cervical dislocation and the testes harvested
following a laparotomy.
Estimation of plasma levels of Testosterone,
Luteinizing hormone and follicle stimulating hormone:
Blood was collected by ventricular puncture into
heparinized bottles and centrifuged at 3000 rpm for 10
minutes using a bench centrifuge at 25oC to obtain the
plasma. Testosterone, LH and FSH was assayed using
the ELISA method using kits from Biotec Laboratories
o
Ltd, UK. Plasma samples collected were stored at -20 C.
Epididymal sperm count, motility and morphology
MATERIALS AND METHODS
Chemicals
L-arginine and ketamine were purchased from Sigma
Chemical (St Louis, MO). 15 mg/100 mg body weight larginine was orally administered to the rats via a metal
oro-pharyngeal canula.
Animals
Twenty male Sprague-Dawley rats weighing 150-250 g
were used for the study. The animals were procured from
the Animal House of the College of Medicine, University
of Lagos and acclimatized for three weeks before
commencement of experiment. Animals were housed in
the departmental animal house at temperature of 28 +
o
2 C with 12 hour light/ 12 hour dark cycle. Commercial rat
feed was sourced from the Livestock feeds Ltd, Ikeja and
water provided ad libitum. Animals were randomly divided
into four groups (A – D) consisting of 5 rats per group.
The testes from each rat were carefully exposed and
removed, trimmed free of the epididymis and adjoining
fatty tissues. Caudal part of the epididymis was removed
and placed in a beaker containing 1mL physiological
saline solution whereupon it was minced using a pair of
sharp scissors. It was kept for a few minutes to allow the
spermatozoa swim out into the solution. Sperm count and
motility were determined as described in Saalu et al.
(2008). Semen drops were placed on the slide and two
drops of warm 2.9% sodium citrate were added. The slide
was covered with a cover slip and examined under the
microscope using X40 objective for sperm motility. Sperm
count was done under the microscope using the
improved
Neubauer’s
counting
chamber
(Haemocytometer). The sperm concentration was then
calculated and recorded in million and expresses as (X) x
6
10 /ml, where X is the number of sperm in a 16-celled
square. Sperm morphology was evaluated using the light
microscope at X400 magnification. Caudal sperm were
taken from the original dilution for motility and diluted
1:20 with 10% neutral buffered formalin (Sigma-Aldrich,
Oakville, ON, Canada). Five hundred sperm from the
Duru et al. 779
Figure 1: Histological sections of testis of rats H & E X 400. A-rt shows right cryptorchid testis
seminiferous tubule devoid of spermatogenic cells (arrowed), interstitial spaces filled with
inflammatory cells. A-lt shows contralateral left testis of same group with normal
spermatogenic cells in seminiferous tubules. B shows normal histo-architectural patterns of
testis of group B rats. C-rt shows right cryptorchid testis with few basal spermatogonia
(arrowed).
sample were scored for morphological abnormalities
(Atessahin et al., 2006). Briefly, in wet preparations using
phase-contrast optics, spermatozoa were categorized. In
this study, a spermatozoon was considered abnormal
morphologically if it had one or more of the following
features: rudimentary tail, round head and detached head
and was expressed as a percentage of morphologically
normal sperm.
Statistical analysis
Data was analyzed using the Student’s T-test and
expressed as mean + SD. P value of p<0.05 was
considered significant.
RESULTS
Histological results
Enzyme assay
Testicular tissue was homogenized in Teflon-glass
homogenizer with appropriate volume of phosphate
buffer 0.1M, pH 7. The samples were then centrifuged at
3500 rpm for 20 minutes in a refrigerated centrifuge. The
supernatant was collected and used for determination of
activities of superoxide dismutase (SOD), catalase,
glutathione reductase and malondialdehyde (MDA). SOD
was determined as described in Sun and Zigma (1978),
catalase activity determined according to the method of
Aebi (1983). Reduced glutathione was estimated
according to the method of Sedlak and Lindsay (1968).
MDA was determined using the method of Buege and
Aust (1978).
Group A: There was necrosis of the spermatogenic cells
in the right cryptorchid testis and inflammation in the
interstitium. The contralateral left testis was normal in
histoarchitecture (Figure 1).
Group B: There was normal histological architecture of
the seminiferous epithelium and interstitial spaces were
essentially normal.
Group C: The right cryptorchid testis showed depletion
of spermatogenic cells in the seminiferous tubules with
mild inflammation in the interstitial spaces. The left
contralateral
testis
showed
normal
histological
appearance (Figure 2).
Group D: Testicular sections of control rats showed 3-5
cell thickness of spermatogenic cells in the seminiferous
780 J. Med. Med. Sci.
Figure 2: Histological sections of testis of rats H & E X 400. C-lt shows contralateral left
testis of group C with normal seminiferous epithelial lining similar to control group D.
Table 1: Seminal fluid analysis of treatment groups
Groups (n)
Treatment
A
Cryptorchid + Larginine
L-arginine
Cryptorchid only
DW
B
C
D
Sperm count
(x106/ml)
15.00 + 8.28α
Sperm motility (%)
50.60 + 26.74α
Sperm morphology
(%)
16.04 + 9.24
15.20 + 9.34α
α
11.50 + 4.53
26.30 + 2.59
59.60 + 25.00
57.00 + 11.60α
87.60 + 8.62
14.30 + 3.60α
15.50 + 3.67α
7.00 + 2.65
Values expressed as Mean + SD; α: P< 0.05; n=5; DW is distilled water
Table 2: Results showing hormonal levels in treated groups
Groups (n)
TT (nmol/L)
LH (IU/L)
FSH (IU/L)
A
B
C
D
7.52 + 2.10
10.72 + 2.68α
4.47 + 0.17α
5.90 + 0.22
0.18 +
0.96 +
0.54 +
0.30 +
0.50 +
0.52 +
0.04 +
0.53 +
0.11
0.13α
0.12α
0.06
0.06
0.07
0.01α
0.26
Values expressed as Mean + SD; α: P< 0.05; n=5; TT is testosterone; LH is luteinizing hormone; FSH is follicle stimulating
hormone
tubules with interstitial spaces that were essentially
normal.
Significant decline in sperm count and motility (p<0.05)
was observed in all treated groups when compared with
control but this was maximal in group C. Similarly, sperm
morphological abnormalities (round head, double head,
no tail) was significantly higher (Table 1) in groups B and
C compared with control.
TT was significantly increased in group B and declined
in group C while group A value was high but not
significant when compared with control. Similar trend was
observed in LH values as well. FSH levels were
maximally depressed in group C whereas the other
groups were not significantly affected (Table 2).
MDA: A significant increase in MDA levels was
recorded in groups A and C when compared with the
control (Table 3).
SOD: A significant decline was recorded in the activity
of SOD in groups A and C compared with control.
Though the value for group B was high, it was not
significant (Table 3).
CAT: Catalase activity declined significantly in group C
while group A was not significant (Table 3).
GSH: Groups A and C recorded a significant decline in
GSH when compared with control (Table 3).
DISCUSSION
The significant decline in sperm count in cryptorchid rats
as well as those that received L-arginine supplementation
is an indication that unilateral cryptorchidism with larginine
supplementation
severely
impaired
spermatogenesis (Ahotupa and Huhtaniemi, 1992).
Duru et al. 781
Table 3: Biochemical parameters assayed in the treatment groups
Group (n=5)
A
B
C
D
MDA (µmol/mg
protein)
0.14 + 0.019α
0.01 + 0.003
α
0.06 + 0.014
0.01 + 0.001
SOD (µ/mg
protein)
3.3 + 0.76α
5.9 + 0.99
α
2.26 + 0.79
4.72 + 0.69
CAT (µ/mg
protein)
16.99 + 2.85
27.24 + 4.59
α
10.42 + 3.65
23.13 + 3.23
GSH (µmol/mg
protein)
0.69 + 0.08α
2.67 + 0.65
α
0.66 + 0.19
2.96 + 0.33
Values expressed as Mean + SD; α: P< 0.05; n=5
Necrosis of spermatogenic cells in the seminiferous
tubules, seminiferous tubular atrophy as well as
interstitial inflammation was observed in the histological
sections of cryptorchid testis after L-arginine treatment
and in testes of cryptorchid rats. Elevated abdominal
temperatures as a result of the experimentally induced
cryptorchidism may have been responsible for this
observation as retention of the testes inside the abdomen
results in disruption of spermatogenesis (Zhang et al.,
2002). L-arginine treatment exacerbated the histoarchitectural
disruptions
following
experimental
cryptorchidism in this study. Previous study by Ozokutan
et al. (2000) had showed that in mice pre-treated with Nmono methyl L-arginine, which acts by inhibiting NO
synthesis, adverse histological effects were absent.
L-arginine is a precursor of nitric oxide, a free radical
(Bechman and Koppenol, 1996) which is reported to
cause decreases in antioxidant capacity and germ cell
apoptosis seen in cryptorchid testes (Chandral., 2009).
This is observed by the decline in sperm count in groups
A and B that had L-arginine supplementation and Larginine alone respectively. NO could possibly have
induced the cytotoxic effects on the testes of these rats.
Again, spermatozoa motility is a high energy-dependent
process and NO have been shown to inhibit the ATP
levels by inhibiting the enzymes involved in electron
transport (Dimmeler et al., 1992). Hence, the low sperm
motility observed in L-arginine treated rats could be due
to oxidative stress-induced damage via NO production
following L-arginine treatment. Cryptorchid rats equally
had low sperm motility which is in agreement with
previous studies by Grasso et al. (1991). Abnormal
sperm rates seen in L-arginine treatment further alludes
to oxidative stress-induced damage while cryptorchidism
in rats have been observed to induce similar aberrations
as reported by Jones et al. (1977). That the epididymis of
the cryptorchid testes had significantly lower sperm
parameters than the unmanipulated testes confirms the
deleterious effect of cryptorchidism on testicular
functions.
Testosterone synthesis is primarily from the Leydig
cells in the interstitium. LH is the major trophic stimulus
for the synthesis of TT. L-arginine treated rats showed
high levels of LH and TT whereas in cryptorchid rats TT
was significantly depressed. The reason for this could be
attributed to heat stress induced by cryptorchidism (Yin et
al., 1997). Kostic et al., 1998 previously reported that a
short immobilization stress in rats produced a sharp
decline in serum testosterone levels and blocked the LHinduced TT production. Also it is possible that the failure
of the negative feedback mechanism resulting from
Leydig cell failure to produce optimal TT could be
responsible for this observation as supported by Saalu et
al., 2007). This suggests that although all cell types of
the testes were affected by cryptorchidism, the most
prominent damage occurred in the seminiferous
epithelium. Thus the Leydig cells, which secrete
testosterone, were minimally affected by experimental
cryptorchidism. The significant decline in FSH levels in
cryptorchid rats could be attributed to the hypo-function of
Sertoli cells in these rats (Au et al., 1983).
The lack of cytoplasm in mature spermatozoa causes
deficiency in antioxidant defense system which is
compensated by the enzymatic and non-enzymatic
antioxidants abundant in seminal fluid. However, the
plasma membrane of spermatozoa is rich in PUFA which
are susceptible to free radical damage and consequent
loss of membrane integrity (Suzuki and Sofikitis, 1999).
From our study, L-arginine treatment did not attenuate
the
lipo-peroxidation
following
experimental
cryptorchidism in rats. MDA levels in groups A and C
were significantly high. Decline in antioxidant enzymes
such as superoxide dismutase, catalase and glutathione
reductase which are markers of oxidative stress (Kehinde
et al., 2005) were significantly altered to the negative in
cryptorchid rats and treatment with L-arginine did not
reverse nor protect the testis from oxidative damage. It is
possible that the increased oxidative stress (high MDA
levels) may be responsible for the depression of these
antioxidant enzymes or alternatively, the inherent
decreased expression of these enzymes may be
responsible for the increased oxidative stress (Sikka et
al., 1995). The result is increased lipid peroxidation,
decreased spermatozoa motility, function and ultimately
infertility.
CONCLUSION
In conclusion, our study demonstrated the detrimental
effect of experimental cryptorchidism on testicular
782 J. Med. Med. Sci.
function and also showed the capacity of L-arginine in
augmenting these deleterious effects.
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