the substrate of nitric oxide synthase, inhibits fertility of male
D. Ratnasooriya, M. G. Dharmasiri
of Zoology, University of Colombo, Colombo 3, Sri Lanka
Asian J Androl 2001 Jun; 3: 97-103
Aim: To examine the effect of L-arginine, the substrate of nitric oxide (NO) synthase, on reproductive function of male rats. Methods: Male rats were gavaged with either L-arginine (100 or 200 mgkg-1d-1), D-arginine (200 mgkg-1d-1) or vehicle (0.9% NaCl) for seven consecutive days. Their sexual behaviour and fertility were evaluated using receptive females. Results: L-arginine (200 mg/kg) had no significant effect on sexual competence (in terms of sexual arousal, libido, sexual vigour and sexual performance). In mating experiments, the higher dose of L-arginine effectively and reversibly inhibited fertility, whilst the lower dose and the inactive stereoisomer D-arginine had no significant effect. The antifertility effect caused by L-arginine was due to a profound elevation in the preimplantation loss mediated possibly by impairment in epididymal sperm maturation, hyperactivated sperm motility and sperm capacitation. Conclusion: Elevated NO production may be detrimental to male fertility.
is now well established that nitric oxide (NO) plays a vital role in the
regulation of male reproductive function[1,2].
NO is formed from the amino acid L-arginine
and the enzyme responsible for its catalysis is nitric oxide synthase
(NOS), which is widely distributed in the male reproductive tract[2,3].
An inhibitor of NOS, N-nitro-L-arginine methyl ester (L-NAME),
inhibits penile erection in rat and man[4,5], reduces human
sperm hyperactivation, impairs GnRH secretion in rat,
and impairs male sexual behaviour[7,8],
libido and fertility of rats.
2 Materials and methods
crossbred albino rats of proven fertility (males weighing 250-300 g and
females, 200-225 g) were used.
All rats were kept in plastic cages under standardised animal house
conditions (temperature: 28-31;
photoperiod: approximately 12 h natural light per day; relative humidity:
50-55%) with free access to pelleted
food (Vet House Ltd., Colombo, Sri Lanka) and tap water.
and D-arginine (Sigma Chemical Co. St. Louis, MO, USA), 100 and
200 mg, were dissolved
in 1 mL of isotonic saline (0.9% w/v NaCl) (BDH Chemicals, Poole, UK).
male rats were randomly divided into 4 groups.
The rats in group 1 (n=10) were orally administered with
and group 2 (n=12) with 200 mgkg-1d-1 of
L-arginine, and the rats in group 3 (n= 6) with 200 mgkg-1d-1
of D-arginine all at 12.00 for 7 consecutive days. Animals in group
4 (n=12) were treated with a similar amount of vehicle.
every dosing, rats were continuously observed for 1 h for clinical signs of
acute toxicity (salivation, rhinorrhoea, lachrymation, ptosis), stress
(erection of fur,exopthalmia), and changes in behaviour.
In addition, during the study period the food and water intake,
the consistency of faeces and the colour of urine were noted daily, and
the rectal temperature determined using a clinical thermometer(TM-II,
Normal glass, Forcal Corporation Tokyo, Japan) once a day during the 7-day
Effect on fertility
rats were individually paired (at 16.00-16.30 h) overnight with a pro-oestrous
female on days 1, 3 and 7 of treatment and day 7 post-treatment.
The pre-coital sexual behaviour (chasing, nosing, anogenital sniffing,
genital grooming, attempted clasping and mounting) of the paired rats
was observed for 1-2 h right after
smears of the females were taken in the following morning (at 8.00-8.30
h) and examined microscopically (100).
If spermatozoa were present, their numbers were determined using
an improved Neubauer haemocytometer (Fison Scientific Equipments, Loughborough,
UK) and gross morphology was observed.
The motility of the spermatozoa was assessed using an arbitrary
scale (0=immotile, 1=low motility, 2=moderate motility, 3=high motility).
If spermatozoa were absent then daily vaginal smearing was undertaken
for at least 8-10 days to
determine the appearance of pregnancy or pseudopregnancy.
At 14 days after mating, the females were subjected to uterotomy
under ether (Fluka, Buchs, Switzerland) anaesthesia to determine the number,
viability and the size of fetuses, and the number of resorption sites.
The gross appearance of corpora lutea was also recorded.
The following reproductive indices were computed: index of libido=(number
mated/number paired) 100, quantal pregnancy=(number pregnant/number
mated) 100, fertility index=(number pregnant/number paired) 100, implantation
index=(total number of implantation/number mated) 100, pre-implantation loss=(number
of corpora lutea-number of implants)/number of corpora lutea
100, post-implantation loss=(number
of implants-number of viable implants)/number of imlpants
Effect on blood counts
day 1 post-treatment, blood was drawn from the tail of rats in groups
2 and 3 (used in 2.5)
under aseptic precautions and their red blood cell (RBC), white blood
cell (WBC) and differential white blood cell (DC) counts were determined
as described by Ghai.
Effect on liver function
collected under 2.6 was allowed to clot at room temperature (28-30)
for 15 min; centrifuged using a Wifug Lab Centrifuge (Eltex of Sweden
Ltd., Bradford, UK) at 3200g for 10 min, and sera was separated.
The serum activities of aspartate
transaminase (EC 126.96.36.199, AST), alanine transaminase (EC 188.8.131.52., ALT),
-glutamyl transferase (EC 184.108.40.206, -GT) and serum concentrations of
albumin and total proteins were determined using test kits (Randox Laboratories
Ltd, Co. Antrim, UK).
Effect on sexual accessory glands
male rats were randomly divided into 2 groups.
The rats in group 1 (n=9) were orally treated with 200 mgkg-1d-1
of L-arginine and the other group (n=6), with the vehicle
for seven consecutive days.
On day 1 post-treatment, these rats were sacrificed with ether
and their body weights recorded.
The weights of the left testis, epididymis and vas deferens, paired
seminal vesicles together with the coagulation glands and lateral prostrate
glands were recorded, and expressed as a percentage of body weight.
The length (pole-pole) of the testes and the maximum
breadth were measured using a vernier caliper.
The pH of the seminal vesicular fluid was determined with narrow
range pH paper (BDH, Poole, UK).
Effect on epididymal sperm counts, motility and gross morphology
cauda epididymis, caput plus corpus epididymis and vas deferens of rats
used in 2.8 were homogenised separately in a ground glass homogenizer
and diluted with a known volume of normal saline; the number of spermatozoa
present in the suspension was determined in duplicate using a haemocytometer
and the sperm counts per
gram of tissue was calculated.
The spermatozoa in the right cauda epididymides were extruded into
a definite amount of isotonic saline and the number of spermatozoa and
the percentage motility were counted under a microscope.
The gross morphology of spermatozoa was also observed.
Effect on histopathology of testes, epididymides and vas deferens
right testis, epididymis and vas deferens of the rats used in 2.9 were
fixed in Bouin's solution, embedded in paraffin wax, sectioned using a
microtome (Feather Safety Razor Co. Ltd, Osaka, Japan), stained with haematoxyline
and eosine, and were examined microscopically.
The thickness of the endothelial lining of the cauda epididymis,
the diameters of the epididymal tubular lumen and the seminiferous tubular
lumen were determined using an eyepiece graticule (Olympus Optical Co
Ltd, Tokyo, Japan).
Effect on hyperactivated sperm motility
effect of L-arginine on sperm hyperactivation was studied in
vitro using BWW
medium as described by Morton et al.
Briefly, sperm (3105/mL) samples collected from the
cauda epididymides of six adult male rats under ether anaesthesia were
incubated for 4 h at 37
in an atmosphere of 5% CO2 and 95% air with different concentrations
of L-arginine (0, 100, 250, 500, or 750 g/mL).
The total number of spermatozoa with normal motility and those
showing hyperactivated motility
(whip lash movement) were counted under a phase contrast microscope (Olympus
Optical Co Ltd, Tokyo, Japan).
Then, the percentage of hyperactivated spermatozoa was calculated.
Effect on contraction of isolated testicular capsule
rats were anaesthetised with ether and their testes removed and placed
in petri dishes containing fresh aerated (95 % O2: 5% CO2)
solution having the following composition (mmol/L): Na+ 143,
5.8, Ca2+ 2.6, Mg2+ 1.2, Cl- 128,
H2PO4- 1.2, HCO3-
25, SO42- 1.2 and glucose 11.1 at pH 7.4.
Capsulotomy was performed carefully under dissecting microscope
(Olympus Optical Co. Ltd., Tokyo, Japan) and suspended in an organ bath
(Rikedenki kogyo Co. Ltd., Tokyo, Japan) at 371under
a resting tension of 2.0 g and allowed to equilibrate for 30 min with
repeated washings. Six
concentrations of L-arginine (50, 75,100,
125, 150 or 200 g/mL) were added cumulatively to the organ bath at 10
min intervals and contractile responses were recorded isometrically using
an isometric sensor (Model IM 20 BS, Star Medicals, Tokyo, Japan) and
displayed on a Natume KN-260 pen recorder (Rikedenki kogyo Co. Ltd., Tokyo,
Effect on contraction of isolated cauda epididymal tubules
epididymides of rats used in 2.12 were removed and placed in Krebs
Henseleit solution. 1.5-2.0
cm portions of the tubule were cut carefully from the vasal end and suspended
in a 50 mL organ bath at a resting tension of 0.5 g at 371.
Following 30 min equilibration period with repeated washing, six
concentrations of L-arginine (25, 50, 75, 100, 125, 150 g/mL)
were added cumulatively
at 10 min intervals and contractile responses recorded isometrically.
Effect on sexual behaviour
male rats were randomly divided into two groups of 12 animals each.
The rats in group 1 were gavaged with 200 mg/kg L-arginine
at 12.00 h and the
other with the vehicle.
At 5 h post-treatment, male rats were individually paired with
a female rat brought into oestrus by subcutaneous injection of estradiol
benzoate (Sigma Chemical Company, St. Louis, MO, USA) and 48 h later by
progesterone (Sigma Chemical Company, St. Louis, MO, USA).
The pre-coital and coital behaviour were observed for 15 min or
until ejaculation. During
this period, the mount latency, the intromission latency, the ejaculation
latency, the number of mounts and the number of intromissions displayed
by the rats were recorded.
Using these parameters the percentage of rats mounted, intromitted,
and ejaculated, and the intercopulatory interval, the copulatory efficiency
and the intromission ratio were calculated.
Effect on muscle strength and co-ordination
male rats were randomLy divided into two groups, and to the first group 200
mgkg-1d-1 L-arginine was gavaged for
seven consecutive days,
while to the second group, the vehicle was given.
Three hours after the last dose, the rats were subjected to bar
holding test to determine the muscle strength, and the
latency to fall in seconds was recorded.
Immediately after this, the rats were subjected to the Bridge test
to evaluate muscle coordination, and the latency to slide
off was recorded in seconds.
L-arginine treatment was well tolerated: there were neither overt
signs of toxicity
and stress, nor gross behavioral abnormalities.
In the treated rats, the food and water intake, the body weight
and the rectal temperature were not significantly different from those
of the controls (data not shown).
the lower (100 mgkg-1d-1) nor the higher (200
mgkg-1d-1) dose of L-arginine induced
marked changes in the pre-coital sexual behaviour as compared to the controls.
Other fertility data are summarised in Table 1.
None of the reproductive parameters tested was significantly altered,
both with the lower dose of L-arginine and D-arginine.
In contrast, with the higher dose of L-arginine, quantal
pregnancy (day 1, by 56%; day 3, by 57% and day 7 by 57%), number of implants
(day 1, by 82%; day 3, by 52% and day 7, by 61%), implantation index (day
1, by 80%; day 3, by 52% and day 7, by 56%) and fertility index (day 1,
by 89%; day 3, by
56% and 57%) were significantly reduced, and the pre-implantation loss
was significantly enhanced (day 1, by 738%; day 3, by 385% and day 7,
by 617%). Seven days
after cessation of the treatment,
these parameters returned or closed to
normal. There was
no post-implantation loss.
WBC, RBC and DC counts of the treated rats were not significantly changed (data not shown). Further, WBC morphology remained to be typical for rats of this age, sex and strain.
with the higher dose of L-arginine for 7 days did not significantly
affect the serum AST, ALT and -GT activities and total protein and albumin
levels (data not shown).
Sexual accessory organs
with the higher dose of L-arginine for 7 days did not significantly
affect the gross appearance and wet weights of the reproductive organs,
as well as the length and width of testes and the pH of the seminal vesicle
and coagulation gland secretions.
Epididymal sperm count and motility
treatment caused a significant (P<0.01) epididymal hyperspermia
(caput+corpus by 149% and cauda by 189%) (Table 2).
However, the motility of the cauda epididymal sperm (68.51.6%
vs 75.40.3% control) and their gross morphology remained unaltered.
histopathology of the testis and vas deferens of treated rats was essentially
similar to those of the controls.
However, the thickness of the caudaepididymal endothelium was significantly
reduced compared with the controls (15.50.5 vs 17.70.5 m,
there were no si
Hyperactivated sperm motility
results of the sperm hyperactivation study are summarised in Table 3.
L-arginine inhibited hyperactivated sperm motility dose-dependently
(r2=0.82, P<0.01) with an EC50 of 203.8
Contraction of testicular capsule
did not induce either contraction or relaxation of isolated testicular
Contraction of cauda epididymal tubules
did not induce either tonic or phasic contraction of epididymal tubules.
Sexual behaviour study
of the male sexual behaviour parameters investigated was significantly
changed in the L-arginine treated rats compared to the controls
(data not shown).
Muscle strength and co-ordination
Table 1. Effects of oral administration of different doses of L-arginine and D\|arginine on some fertility parameters of male rats (meanSEM, ranges in parentheses).
bP<0.05, cP<0.01, compared with controls (Mann-Whitney U-test, G-test).Table 2. Effects of oral administration of 200 mgkg-1d-1 of L-arginine for seven days on the distribution of sperm in the reproductive tract of rats (meanSEM, range in parenthesis).
3. Effect of different
concentrations of L-arginine on the hyperactvation
of spermatozoa in vitro (meanSEM, n=6, range in
This study demonstrated that oral administration of L-arginine had no effect whatsoever on sexual competence of male rats: sexual arousal (in terms of latency to mount, intromit or ejaculate), libido (judged by % mounting, intromitting or ejaculating in the behavioral study and pre-coital sexual behaviour and index of libido in the fertility study), sexual vigour (determined by mounting-and-intromission frequency and copulatory efficiency) or sexual performance (judged by intercopulatory interval) remained unchanged. The doses of L-arginine used here were similar to those in in vivo studies to reverse the effects of L-NAME[15,16] and higher than that used to promote NO synthesis in vitro. Hence, it is presumed that the doses of L-arginine used in this study were capable of elevating the NO levels in the brain and the reproductive organs. Male sexual behaviour of rat is inhibited by L-NAME[7,8] and this effect has been attributed to reduction of NO at brain centres controlling reproduction. Thus, the inability of L-arginine to affect sexual behaviour suggests that a thresh old level of NO exists in brain for the regulation of male sexual function. Alternatively, the inability of L-arginine to impair sexual competence may be due to differences in the NO synthetic activity of NOS isoforms.
profoundly inhibited the fertility. In contrast, the inactive stereoisomer,
D-arginine, had no effect whatsoever on fertility.
Thus, the antifertlity effect of L-arginine can be attributed
to elevated NO production.
The onset of
this antifertility effect was very rapid (within hours), lasted throughout
the treatment period (days 1-7) and was rapidly reversible (within 7 days)
on cessation of treatment.
Further, L-arginine did not induce marked changes in testicular
size or wet weight and in the histopathology of the seminiferous tubules.
These observations collectively suggest an extragonadal site of
action. There were
no overt signs of general toxicity, haemotoxicity, hepatotoxicity, stress, sedation,
behavioural and postural abnormalities, motor incoordination or muscle
deficiency in treated rats.
Thus, the antifertility action is not attributable to its possible
toxic effect. Further,
the antifertility action is unlikely to be mediated via a change in testosterone
level since there was no reduction in wet
weights of sexual accessory glands or pH of seminal vesicle fluid.
In rats, a minimum number of intromissions of sufficient strength
is essential for a female to become pregnant.
L-arginine treatment did not inhibit intromission frequency
or induce erectile dysfunction.
Therefore, impairment of fertility cannot be attributed to such
a mechanism. Similar
pharmacological action has been reported for L-NAME, a drug that
reduces NO level.
L-arginine-induced antifertility effect was accompanied
with an enhancement of pre-implantation
loss as with L-glycine, another amino acid. This
pre-implantation loss was not due to a reduction in sperm numbers in the
ejaculate, nor was it due to the presence of morphologically defective
sperm in the ejaculate.
Further, the enhanced pre-implantation loss is unlikely to be due
to an impairment of sperm transport in the oviduct, as the motility of
both cauda epididymis and ejaculated sperm was uninhibited by L-arginine.
The epididymal sperm count was increased by the L-arginine
treatment, indicating a shorter epididymal transit time.
However, organ bath study indicates that the epididymal hyperspermia
was unlikely to be due to L-arginine-induced increase in contraction
of the testicular capsules or epididymal tubules.
Such an action could cause an interruption of sperm maturation
process resulting in ejaculates with higher number of sperm of low fertilizing
ability. In contrast,
an impairment in fertilising potential may have resulted from disrupted
epididymal function: L-arginine induced thinning
of epididymal endothelium.
Impaired fertilizing potential enhances pre-implantation loss.
also caused a dose-dependent impairment of hyperactivated motility in
vitro, which can obviously inhibit fertilizing potential of sperm,
thereby inducing a pre-implantation loss.
Hyperactivated sperm motility is essential for the
passage of sperm through zona pellucida.
An impairment of hyperactivation also indicates an inhibition of
sperm capacitation. An inhibitory effect on capacitation may by itself
potentiate pre-implantation loss.
However, in this study, it is uncertain whether the defective sperm
function is originated by a direct action of L-arginine on sperm
(as NOS is present in sperm) or an indirect action via
epididymis (as there was a reduction in the endothelial lining) or by
both mechanisms. Hence, further studies are warranted to elucidate the
precise mechanism of arginine-induced defective sperm function.
Forstermann V, Gath I, Schwarz P, Closs EI, Kleinert H.
Isoforms of nitric oxide
synthase, properties, cellular distribution and expressional control.
1995; 50: 1321-32.
W. D. Ratnasooriya, Department of Zoology, University
of Colombo, Colombo 3, Sri Lanka.