J Physiol Biochem, 64 (3),231-240,2008

Inhibitory effects of la, 25dihydroxyvitamin

D3
and Ajuga iva extract on oxidative stress, toxicity
and hypo-fertility in diabetic rat testes
K. Harndenl, s. Carreau/, K. jarnoussr', F. Ayadi ', F. Garrnazi",
N. Mezgenni" and A. Elfeki!
1Animal Ecophysiology,
Faculry of Seienees, Sfax, Tunisia. 2USC 2006 INRA- EA 2608,
Biochemistry-University
of Caen, Franee. 3Biochemistry laboratory, CHU H. Bourguiba, Sfax, Tunisia. "Radio-immunology
laboratory, CHU H. Bourguiba, Sfax, Tunisia.

(Received on May, 2008)

K. HAMDEN,
S. CARREAU,
K. JAMOUSSI,
F. AYADI, F. GARMAZI,
N. MEZGENNI
and A. ELFEKI. Inhibitory effects of 1 a, 25dihydroxyvitamin DJ
and Ajuga iva extract on oxidative stress, toxicity and hypo-fertility in diabetic rat
testes. J Physiol Bioehem, 64 (3), 231-240, 2008.
The aim of the eurrent study is to investigate the therapeutic and preventive effeets
of la, 25dihydroxyvitaminD3
(1,25 (OHh D3) and Ajuga iva (Al) extraet on diabetes toxicity in rats testes. Thus diabetic rats were treated with la, 25dihydroxyvitaminD3 or Ajuga iva extract as both therapeutie and preventive treatments on diabetes toxicity in rats testes. Our results showed that diabetes indueed a deerease in
testosterone and 17~-estradiolleve!s
in testes and plasma. Besides, a fall in testieular
antioxidant capacity appeared by a deerease in both antioxidant (superoxide dismutase (SOD), eatalase (CAT) and glutathione peroxidase (GPx) activities) and nonenzymatie antioxidant (copper (Cu), magnesium (Mg) and iron (Fe) leve!s). All theses ehanges enhaneed testicular toxicity (inerease in testicular aspartate amino
transaminase
(AST), alanine amino transaminase
(AL T), laetate dehydrogenase
(LDH) activities and the lipid peroxidation and triglyeeride (TG) leve!s). In addition,
a deerease in testieular total cholesterol (TCh) leve! was observed in diabetic rats
testes. Al! the ehanges lead to a decrease in the total number and mobility of epididymal spermatozoa. The administration
of 1a,25dihydroxyvitaminD3
and Ajuga
iva extraet three weeks before and after diabetes induetion interfered and prevented
diabetes toxieity in the reproduetive system. 1,25 (OH)2 D3 and Ajuga iva extraet
blunted all ehanges observed in diabetie rats. To sum up, the data suggested that 1,25
(OHh D3 and Ajuga iva extraet have a proteetive effeet on alloxan-indueed
damage
in reproductive system by enhancing the testosterone and 17~-estradiolleve!s,
eonsequendy proteeting from oxidative stress, cellular toxieity and maintaining the number and motility of spermatozoids.
Key words: 1,25 (OH)2 D3, Ajuga iva extraet, Diabetes rnellitus, Testosterone.
Correspondence to K. Hamden (Tel. +00 216 97469111; Fax. +00 216 74274437; e-mail: hamdenkhalid@
yahoo.com).

232

K. HAMDEN,

S. CARREAU,

Diabetes mellitus, a major endocrine

disorder and a growing health problem in
most countries, is now emerging as a
deadly disease. Three quarters
of the
world's 400 million adults with diabetes
are in western countries and the disease is
also gradually
becoming
prevalent
in
developing countries (24). Hyperglycemia
leads to long-term complications of diabetes, which are the major causes of morbidity and mortality in human populations. (24).
Chronic elevation of blood glucose,
mostly often with no symptoms present
to alert the individual to the presence of
diabetes, will eventually lead to tissue
damage and consequently serious disease
in many organ systems such as the kidneys, liver and vascular system (6, 7, 9,
11). Also hyperglycemia lead to the dysfunction of the reproductive
system in
men and rats (2, 19). Diabetic rats exhibit
testicular dysfunction including a decrease
in Leydig, Sertoli and sperm count (3, 4,
25). Moreover,
low 17~-estradiol
and
testosterone
levels and impaired spermatogenesis
are characteristics
of the
testis of diabetic animals (4, 25). Diabetesrelated effects on testicular function have
been attributed to the lack of antioxidant
capacity and oxidative stress which is
believed to affect the development of diabetic-associated dysfunction. The balance
between oxidant and antioxidant species
has been proposed to have an important
rol e in the emergence of diabetic eomplications.
The dietary factors playa key role to in
the prevention and cure of various human
diseases associated to oxidative stress,
including diabetes, cancer and aging (13,
14, 22, 33). Steroids molecules such as
estrogens, vitamin D3 and phytoestrogens
continue to provide valuable therapeutic
agents, in both modern medicine and in
J Physiol Biochem, 64 (3), 2008

K. JAMOUSSI,

F. A YADI et al.

tradicional systems (18). Steroids bio-molecules provide potential benefits in the

regulation of insulin and glueagon secretion, accelerate pancreas regeneration,
.prevent glucose toxicity, and inhibit the
activity of several enzymes included in
oxidative stress via diabetes and cancer
installation,
including protein tyrosine
kinases, DNA topoisomerase I and DNA
topoisomerase II and ribosomal S6 kinase
(5,31,34).
This experimental
study aimed at
investigating the effects of diabetes on the
number and mobility of epidymal spermatozoa, lipid peroxidation,
enzymatic
and non-enzyrnatic
antioxidants eontents
and cholesterol and triglycerides levels in
testes. In addition, we studied the therapeutic and preventive
effeets of the
administration
of 1,25 (OH)2 D3 and
Ajuga iva extract, a plant extract containing many phytoeedysteroids
(35), on the
reproductive system in diabetic rats.

Material and Methods

Animals.- Male Wistar rats, with body
weights of 180 to 200 g and bred in the
Central Animal House and obtained from
the Central Pharmaey, Tunisia, were used
in this study. The animals were fed on a
pellet diet (Soceo, Sfax, Tunisia) and water
ad libitum. The animals were maintained
in a controlled environment under standard conditions
of temperature
and
humidity with an alternating light-anddark cycle. The handling of the animals
was approved by the Tunisian Ethical
Committee for the eare and use of laboratory animals.
Preparation 01 plant extract.- The
aqueous extract was prepared according
to the traditional method deseribed elsewhere (30). Briefly, Ajuga iva was eolleet-

1,25

(OH), D, AND PHYTOECDYSTEROIDS

ed locally and air dried, powdered and

extraeted with double distilled water by
refluxing for 36 hr (12 X 3) at 80 "C. The
extract thus obtained was vacuum evaporated so as to turn it into powder formo
The extraet was re-dissolved in double
distilled water just before administration.
The extraet and vitarnin D3 were administered by oral route.
Experimental
induction 01 diabetes.Rats were i.p. injected three times (one
injeetion per day) with a freshly prepared
solution of alloxan monohydrate
in normal saline at a dose (60 mg/kg bw) for
three successive days. Sinee alloxan is
capable of producing fatal hypoglyeemia
as a result of massive pancreatic insulin
release, rats were treated with 20% glucose solution orally 6 hours after i.p injection of alloxan. The rats were then kept
for the next 24 h on 5% glueose solution
bottles in their eages to prevent hypoglycemia. After 2 weeks, rats developed
moderate diabetes as confirmed by glyeosuria and hyperglyeemia (i.e., with blood
glucose levels of 2.1 to 2.8 gil).
Experimental procedure.- A total of 48
rats (40 diabetic surviving rats and 8 normal rats) were used and they were divided
into six groups with 8 rats per group:
Group 1, normal untreated rats (control);
Group 2, diabetie rats (Diab); Group 3
and 5, therapeutic effeet: received Ajuga
iva aqueous extract (50mg/Kg bw daily)
(Group Al, ther) (16) or (5000lU/kg bw
daily) 1,25 (OH)2 D3 (Group VD, ther)
(36) by gastric gavage for three weeks
after alloxan injection; and Group 4 and 6,
preventive effect: received the Ajuga iva
aqueous extraet (Group Al, prev) or 1,25
(OHh D3 (Group VD, prev) by gastrie
gavage for three weeks before alloxan
injeetion.
J Physiol Biochem, 64 (3), 2008

D;

DLUlETlC ~

,,-=--=-=-=-=5

me

After rwo months of

S-12rt o: ::::e
experimentation,
the animals were sa ..
fieed by decapitation, and the trunk blood
collected. The serum was prepared by
eentrifugation (1500g, 15 min, 4 "C) and
the testis were removed and cleaned of fati
all these samples were sto red at -80C
unti] used.
Epididymal sperm count.- Spermatozoa
were collected from an equallength of the
cauda epididymis of each rat by flushing
with the same volume (10 mL) of a medium eontaining 140 mmollL NaCl, 0.3
mrnol/L KCI, 0.8 mmol/L Na2HP04,
0.2
mmol/L KH2P04
and 1.5 mmollL Dglueose (pH 7.3). The collected samples
were centrifuged at 100 x g for 2 min, and
the pellets were re-suspended in 10 mL of
fresh medium. An aliquot (100 p l.) was
mixed with an equal volume of 1% Trypan blue, then the number of spermatozoa
and the motility were determined (3).
Analytical methods.- After the homogenization of the testes in a phosphate
buffer (1 g/2 mi), steroids were extracted
by diethylether aecording to our reported
method (17). The 17f3-estradiollevel
was
then measured by RlA using highly speeifie antibodies
from P.A.R.I.S (Paris,
Cornpiegne, France). The intra- and interassay eoefficients of variation were 8%
and 5%. The testosterone level was then
measured by RlA using highly specific
antibodies from P.A.R.I.S (Compiegne,
France). The intra- and inter-assay coefficients of variation were 4.6% and 7.5%.
The lipid peroxidation
in the testes of
control and all treated groups of animals
was measured by the quantification
of
thiobarbituric
acid reactive substances
(TBARS) as deseribed (8). The activity of
SOD was assayed speetrophotometrieally
and expressed as U/mg protein (26). The

-----------~~~..._-

-----

234

K. HAMDEN,

S. CARREAU, K. ]AMOUSSI,

GPx actrvrty was measured as described

(28)
and
expressed
as
prnoles
GSS/(min"mg
protein) (28). The CAT
activity was determined colorimetrically
(1) and expressed as umoles f H202 consumed/ (rnin+mg protein). Total protein
was determined by the method of LOWRY
using bovine serum albumin as the standard (23). The activity of LDH, AST,
ALT and the level of Mg, Cu, Fe, TCh
and TG in testes were measured using
commercial kits from Biomagreb, Tunis,
Tunisia and Biomerieux, Lyon, France.

###

~
E

2.5

-=-

Qj

1:

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Qj
1.5
Vi
o
Vi

**##
**#

**#

2
E
::l

0.5

VI

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o'"

<:flJ>
..:,,<:)'

(j

0"'-

~0'

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..:"Q'

~",' qfP"'i>' i>'

25

Statistical analysis.- Data are presented

as means SD. The determinations were
performed from eight animals per group
and the differences were examined 'by the
one-way analysis of variance (ANOVA)
followed by the Fisher test (Stat View)
and the significance
was accepted
at
p<0.05.

F. AYADI et al.

Ci

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20

* #

* #

-=Qj

"E!Qj
Vi
o
Vi 10
2
15

~
'5
'o"
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***#

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Qj

Results

1-

o
Control

Serum and testicular testosterone and

testicular 17 f3-estradiol levels.- Testicular
and serum testosterone values in control
rats were 20.4 nmol/g and 2.4 nmol/l
respectively
which
was significantly
decreased by 77 and 82% respectively in
diabetic animals. In diabetic rats treated
with 1,25 (OH)2 D3 or Ajuga iva extract,
testosterone
levels were significantly
higher than the observed in untreated diabetic rats. Also diabetes
induced
a
decrease in testicular 17j3-estradiol level
by 41 % and the administration
of both
1,25 (OH)2 D3 or phytosteroids
of Ajuga
iva ameliorated this decrease (Fig. 1).

Testicular SOD, CAT and GPx activities and TBARs levels.- The SOD, CAT
and GPx activities in the untreated diabetic group were significantly lower than that
J Physiol

Biochem,

64 (3), 2008

Diab

VD,
ther

VD,
prev

Al,
ther

Al,
prev

350

rf

300

Ci

-;,
.!::

**#

(5

**

:; 200
E
ViQj 150
;;
'5
o

~
Qj

1-

250

#
**#

100
50
O

~a-

Cp'"
Fig. 1. Serum and testicular testosterone and testicular 17~-estradiollevels after 1,25 (OHh D] or Ajuga
iva extract administration in diabetic rats in preventive and therapeutic experiments.
Values are meanSD (n=8) "P < 0.05, ':-o,p < 0.01, and
"""-P < 0.001 vs. control group. #p < 0.05, ##p < 0.01,
###p < 0.001 vs. diabetic group.

1,25

(OHJ, D3 AND PHYTOECDYSTEROIDS

IN DIABETIC

235

RAT TESTES

14
'2

~ 3.5
C. 3

**##

'"

E
3 2.5
~
.;;
~

1.5

***#

***

***

1.2

***

3,5
3

***#

***#

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**##

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**#

'2

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[

**##
**##
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0.8

*#

*#

.E'"

"O 0.6
E

###

..s
(J)

a::

OA

c(

0.2

o +-,----'---,-

Fig. 2. Enzyme aetivities o[ SOD (U/mg protein), CAT (U1mg protein/min) and GPX (U1mg protein/min)
activities and lipid peroxidation levels (TBARS) (nM/mg protein) in testes in pre/post 1,25 (OHh D3 or Ajuga
iva extraet administration in diabetie rats.
Values and symbols as in Fig. 1.

in the other groups (Fig. 2). However, the

activities of all theses enzymes were significantly increased after administration
of 1,25 (OH)2 D3 or Ajuga iva extract to
diabetic rats (Fig. 2), with the effects more
pronounced
in preventive experiments.
AIso, TBARs values in testes increased in
untreated diabetic group compared to that
in the control group (P < 0.01) and the
administration of 1,25 (OHh D3 or Ajuga
iva extract reduced the levels compared to
the untreated-diabetic
group, and this
J Physiol Biochem,

64 (3), 2008

decrease was more pronounced

tive experiments (Fig. 2).

in preven-

Testicular Mg, Cu, Fe, TCh and TG

values and LHD, AST and ALT activities.- Testicular magnesium, iron, copper
and total cholesterol levels diminished in
diabetic rats (Table 1). After 1,25 (OHh
D3 or Ajuga iva extract administration, a
significant increase in all these values was
observed as compared to untreated di abetic rats. Contrarily, testicular triglyc-

236

K. HAMDEN,

S. CARREAU, K. ]AMOUSSI,

F. AYADI et: al.

Table 1.Testicular AST (U/mg protein), AL T (U/mg protein), LOH (U/mg protein), TCh (mg/g), TG (jJg/g), Mg
(mg/g), Cu (jJg/g) and Fe (jJg/g) contents in diabetic rats in pre/post
administration in diabetic rats.

1,25 (OHh 03 or Ajuga iva extract

Results and symbols as in Fig. 1.

Diabetic

Groups

Control

AST
ALT
LDH
TCh
TG

6.320.2
111.3
5.4 1.3
1.60.36
1.50.13
21.42.6
2.60.6
2.330.2

Fe
Mg
Cu

342.2***
41 2.3***
575***
0.160.02***
2.50.12***
11.022.1 ***
0.550.1 ***
0.430.07***

VDther
13 1 .4***###
19.32.2**###
326.9***###
0.650.1 ***###
1.90.16**###
13.71.5**#
2.070.2##
0.840.05**##

12
IS)

o
...

10

3
IU
o
N
o
16
E

* #

*##
*#
**#

Q.

***

CI)

-;
'O

1-

VDprev
10.90.9*###
16.3 1*###
17 4.6***###
0.680.04***###
1.70.19***
14.9 1.4**#
2.330.1 ###
1.130.10**###

Alther
25.7 0.8***#
30.33.4 ***##
37.7 4.5***##
1.080.04**###
2.10.13*##
14.3 1. 7**#
1.690.2**##
0.820.109**#

Alprev
19.60.7***##
23.8 1.1 ***###
23.89.6***###
1.360.08*###
1.80.11 **#
15.4 1.5**##
2.150.2*###
1.140.110**###

erides levels increased by 40% in diabetic

rats, which was significantly diminished
after 1,25 (OH)2 D3 or Ajuga iva extract
treatment.
A significant increase in the activity of
testicular
LDH, AST and ALT was
observed in diabetic rats testes when compared to non-diabetic rats. After treatment 1,25 (OHh D3 or Ajuga iva, all
these enzyme
activities
significantly
decreased
as compared
to diabetic
untreated rats.

Discussion

IS)

...

3
IU
5
oN

tQ.

E
CI)

.!

**##
**##
**#

***

:co

::
0+-,----'---,---

Fig. 3. Total number of spermatozoa in cauda epididymis and measurement of their motility in pre/
post 1,25 (OHh D3 or Ajuga iva extraet administration in diabetie rats.
Values and symbols as in Fig. 1.

J Physiol Biochem, 64 (3), 2008

Previous results indicate that hyperglycemia induces the nonenzymatic glycation of proteins via Maillard reaction,
resulting in Schiff-base products
and
Amadori products that engender ROS
production (2,25). These ROS attack, specially high in steroid synthesizing tissues,
could lead to a decrease in Leydig and Sertoli cells (4), causing a decline in testosterone and estro gens secretion by testicular cells (17). This breakdown in testicular
and plasmatic testosterone and especially
estrogens levels in diabetic rats is accompanied by excessive oxidative stress and

..'I

'[

'",

1,25

(OH) D, AND PHYTOECDYSTEROIDS

lipid peroxidative cell damage. In this

work, enhanced oxidative stress in the diabetic testes cells is suggested by the significant increase in testicular lipid peroxidation as well as a significant decrease in testicular antioxidants including SOD, CAT
and GPx activities and the los s of enzyme
cofactors, namely copper, magnesium and
iron. It has been reponed (3, 20) that diabetes induces the inhibition of SOD and
CAT activities, increased lipid peroxidation and activation of apoptotic process in
diabetic rat testes. Besides, our results
showed a decrease in testicular cholesterol
levels in alloxan-treated rats thar might be,
in part, attributed to the inhibition of
transpon of cholesterol to testes by the
glycation reaction between glucose and
the oxidation of cholesterol transponer
via LDL and lipoprotein. In addition, the
decrease in testicular cholesterol level
might reflect a direct reaction between
cholesterol or cholesterol transponer and
glucose generated by alloxan. Accordingly, the decrease in the antioxidant capacity and the increase of testicular lipid peroxidation (sign of basic deteriorative
process) in testicular cells caused in
increase in indices of cellular toxicity
(such as AST, ALT and LDH activities)
and a decrease in the count and motility of
spermatozoa in accordance to the effect of
diabetes in human (25).
Several investigators have shown that
1,25 (OH)2 D3 and phytoestrogens content in plants represents a potential antidiabetic activity by various mechanism,
such as the inhibiting of the development
of insulitis, the acceleration of pancreatic
regeneration, the increase in insulin secretion and sensitivity and the enhancement
of the cellular antioxidant defense system
protecting of the breakdown of estrogens
and testosterone levels; hormones essentials in homeostasis regulation (4, 18, 27).
J Physiol Biochem,

64 (3), 2008

IN

DlABETlC RAT TESTES

237

The present data revealed that treatment

of diabetic rats with vitamin D3 or Ajuga
iva extract administration might protects
testes cells from damage and death
induced by free radicals or glucose. These
effects might lead to an increase in plasma
and testicular testosterone and 17~-estradiol
levels
in alloxan-1,25
(OHh
D3/Ajuga iva extract treated rats. The
increase in 17~-estradiol and testosterone
level increases the testicular SOD, CAT
and GPx activities, consequently preventing lipid peroxidation in testicular cells
and maintaining copper, magnesium and
iron concentrations. These results are in
agreement with several studies in humans,
where E2 via testosterone reverses the
effect of menopause
on glucose and
insulin metabolism, resulting an increased
pancreatic insulin secretion as well as
improved insulin resistance (27, 29). In
mice, E2 acting partly through ERa, protects pancreatic ~-cells from apoptosis
induced by oxidative stress (21). Furthermore, estrogens are know for their antioxidant activities (5, 14, 34), thus inhibiting
some changes associated to oxidative
stress such as decrease in the levels of Cu,
Mg and Fe, cofactors of SOD and catalase.
This effect might maintain SOD and catalase activities or prevent their inhibition.
This is in accordance with the findings of
DUZGUNERet al. (12) who correlated the
recovery of SOD activity to the increase
in its cofactor concentrations in diabetic rats. In addition, the administration of
steroid molecules such as estro gens and
flavonoids in aged rats increased the level
of aromatase (enzyme converting testosterone to estradiol in testes) and estro gens
receptors ERa/~ transcript in testes (17).
The increase of testosterone and estradiol
levels, hormones essential for spermatogenesis (lO), in diabetic rats treated with vitamin D3 and Ajuga iva extract can protect

238

K. HAMDEN, S. CARREAU, K. jAMOUSSI,

against the alloxan indueed deerease in the

number and rnotility of spermatozoids.
Aeknowledgements
This work was supported by the Tunisian Ministry of Higher Education and Scientific Research
and Technology and the Tunisian Ministry of Public
Heath. We extend our thanks to Mr Hafedh
BJAOUI, teacher of English at the Sfax Faculty of
Science, for having proofread this paper.
K.
HAMDE
S. CARREAU,
K.
JAMOUSSI,
F. AYADI, F. GARMAZI, N.
MEZGENNI
yA. ELFEKI. Efectos inhibitorios de la 1,25 (OHh Vit. D3 y del extracto de
Ajuga iva sobre el estrs oxidativo y toxicidad
testicular
en ratas diabticas.
J Physiol
Biochem, 64 (3),231-240,2008.
Se investigan los efectos preventivos y teraputicos de la 1,25 (OHh Vit. D3 y del extracto de Ajuga iva sobre la toxicidad diabtica en
testculos de rata. Los resultados mostraron
que la diabetes produce descenso de los niveles
de testosterona
y 17~-estradiol en plasma y
testculo y cada de la capacidad antioxidante
testicular, tanto enzimtica (actividad CAT y
GPx) como no enzimtiea (niveles de Cu, Mg
y Fe). Ello se acompaaba de incremento de
aumento de la actividad AST, ALT y LDH Y
de la peroxidacin lipdica y de descenso del
colesterol testicular. Adems, se observaba disminucin del nmero total y de la movilidad
de los espermatozoides
epididimales. En suma,
la administracin
de 1,25 (OHh Vit. D3 y de
extracto de Ajuga iva amortigua la toxicidad
debida a la diabetes sobre el sistema reproductor, incrementando
los niveles de testosterona
yestradiol.
Palabras clave: 1,25 (OH)2 Vir. D3, Extracto de
Ajuga iva, Diabetes mellitus, Testosterona.

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