ISI Impact Factor (2004): 1.096

Editor-in-Chief
Prof. Yi-Fei WANG,

L-arginine, the substrate of nitric oxide synthase, inhibits fertility of male rats

W. D. Ratnasooriya, M. G. Dharmasiri

Department of Zoology, University of Colombo, Colombo 3, Sri Lanka

Asian J Androl  2001 Jun; 3:  97-103

Keywords: L-arginine; nitric oxide; fertility; libido; sex behavior; pre-implantation loss; sperm motility; contraceptives

Abstract

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 mg·kg^-1·d^-1), D-arginine (200 mg·kg^-1·d^-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.

1 Introduction

It 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^[1] 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^[6], impairs GnRH secretion in rat^[2], and impairs male sexual behaviour^[7,8],  libido^[8] and fertility of rats^[8].

2 Materials and methods

2.1 Animal

Healthy 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. 

2.2 Arginine preparation

L- 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).

2.3 Treatment

Forty male rats were randomly divided into 4 groups.  The rats in group 1 (n=10) were orally administered with 100 mg·kg^-1·d^-1  and group 2 (n=12) with 200 mg·kg^-1·d^-1 of L-arginine, and the rats in group 3 (n= 6) with 200 mg·kg^-1·d^-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.

2.4 Observation

After 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 treatment period.

2.5 Effect on fertility

The 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 pairing.  Vaginal 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］ ×100.

2.6 Effect on blood counts

On 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^[10].

2.7 Effect on liver function

Blood 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 2.6.1.1, AST), alanine transaminase (EC 2.6.1.2., ALT), γ-glutamyl transferase (EC 2.3.2.2, γ-GT) and serum concentrations of albumin and total proteins were determined using test kits (Randox Laboratories Ltd, Co. Antrim, UK).

2.8 Effect on sexual accessory glands

Fifteen male rats were randomly divided into 2 groups.  The rats in group 1 (n=9) were orally treated with 200 mg·kg^-1·d^-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).

2.9 Effect on epididymal sperm counts, motility and gross morphology

The 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^[11].  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^[11].  The gross morphology of spermatozoa was also observed.

2.10 Effect on histopathology of testes, epididymides and vas deferens

The 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).

2.11 Effect on hyperactivated sperm motility

The effect of L-arginine on sperm hyperactivation was studied in vitro using BWW medium as described by Morton et al^[12].  Briefly, sperm (3×10⁵/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 μg/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.

2.12 Effect on contraction of isolated testicular capsule

Six rats were anaesthetised with ether and their testes removed and placed in petri dishes containing fresh aerated (95 % O2: 5% CO₂) Kreb‘s Henseleit solution having the following composition (mmol/L): Na⁺ 143, K⁺  5.8, Ca²⁺ 2.6, Mg²⁺ 1.2, Cl^- 128, H₂PO₄^- 1.2, HCO₃^- 25, SO₄^2- 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 37±1℃under 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 μg/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, Japan).

2.13 Effect on contraction of isolated cauda epididymal tubules

Cauda epididymides of rats used in 2.12 were removed and placed in Kreb’s 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 37±1℃.  Following 30 min equilibration period with repeated washing, six concentrations of L-arginine (25, 50, 75, 100, 125, 150 μg/mL) were added cumulatively at 10 min intervals and contractile responses recorded isometrically. 

2.14 Effect on sexual behaviour

Twenty-four 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^[13].

2.15 Effect on muscle strength and co-ordination

Twelve male rats were randomLy divided into two groups, and to the first group 200 mg·kg^-1·d^-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^[14], and the latency to fall in seconds was recorded.  Immediately after this, the rats were subjected to the Bridge test to evaluate muscle coordination^[14], and the latency to slide off was recorded in seconds^[14].

2.16 Statistical analysis

3 Results

3.1 Adverse effects

The 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). 

3.2 Fertility

Neither the lower (100 mg·kg^-1·d^-1) nor the higher (200 mg·kg^-1·d^-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.

3.3 Blood counts

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.

3.4 Liver function

Treatment with the higher dose of L-arginine for 7 days did not significantly affect the serum AST, ALT and γ-GT activities and total protein and albumin levels (data not shown).

3.5 Sexual accessory organs

Treatment 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.

3.6 Epididymal sperm count and motility

L-arginine 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 (68.5±1.6% vs 75.4±0.3% control) and their gross morphology remained unaltered. 

3.7 Histopathology

The 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 (15.5±0.5 vs 17.7±0.5 μm, P<0.05).  However, there were no si gns of spermatic granuloma formation or disruption of the order of cellular arrangements in the epididymis.

3.8 Hyperactivated sperm motility

The 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 μg/mL.

3.9 Contraction of testicular capsule

L-arginine did not induce either contraction or relaxation of isolated testicular capsules.

3.10 Contraction of cauda epididymal tubules

L-arginine did not induce either tonic or phasic contraction of epididymal tubules.

3.11 Sexual behaviour study

None of the male sexual behaviour parameters investigated was significantly changed in the L-arginine treated rats compared to the controls (data not shown).

3.12 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 (mean±SEM, ranges in parentheses).