The Effects of L-arginine and It's Antagonists L-NAME and Methylene Blue on Sexual Behaviors in Mice
Salma J. Askar, Farid J. Al- Tahan and Mohanad A.Al-Bayati
Department of Physiology and Pharmacology, College of Veterinary
Medicine,University of Baghdad, Iraq
Summary
The present study was done to focus light on possible enhancement of the functional performance of male and female mice sexual activities by using L-arginine as a precursor of nitric oxide (NO). the results showed a significant increase at (P<0.05) in male latency of aggressiveness (second), decrease in frequency of aggressiveness, and decrease in the duration of aggressiveness (second) in L-arginine treated groups and decrease in latency of aggressiveness (second), increase in frequency of aggressiveness, and increase in duration of aggressiveness (second) in both L-NAME and methylene blue treated groups in both periods of treatment 15 and 30 days as compared with control groups. The results of male sexual behaviors showed significant decrease at (P<0.05) in latency of copulation (second), increase in frequency of copulation (per – hour) and increase in duration of copulation (second) in L-arginine treated groups, while there abolishment of sexual behavior parameters in both L-NAME and methylene blue treated groups in both periods of treatment 15 and 30 days as compared with control groups.
The results of female sexual behavior revealed a significant increase at (P< 0.05) in lordosis quotient in L-arginine treated groups and abolished lordosis quotient in both L-NAME and methylene blue treated groups in both periods of treatment 15 and 30 days. The hormonal levels assessment for both genders revealed a significant increase at (P<0.05) in testosterone, FSH, LH, estrogen and progesterone hormonal levels in L-arginine treated groups in male and female mice, respectively in both periods of treatment 15 and 30 days as compared with control groups, while there were decrease in these hormonal levels in both L-NAME and methylene blue in both genders, in both periods of treatment 15 and 30 days as compared with control groups.
It could be concluded that L-arginine – NO pathway plays an important role in an improving and enhancement of sexual activities in both genders.
Introduction
L-arginine is one of modifiers of behaviors; it acts as a neuromodulator in the central and peripheral nervous systems from which nitric oxide (NO) is derived in presence of nitric oxide synthase enzyme (NOS) (1). L-arginine–NO pathway affects both sexual motivation and performance, although much of the effects of nitric oxide on male sexual performance reflect the direct consequences of the well- established effects of nitric oxide on penile erection (2). Treatment of male rats with L-arginine did reduce the latency of first mount and increase the number of mounting attempts of estrous females by male rats (3 and 4).
The stimuli from a receptive female and /or copulation itself leads to the release of dopamine (DA) in at least three integrative hubs: The nigrostriatal system promotes somatomotor activity; the mesolimbic system subserves numerous types of motivation; and the median preoptic area (MPOA) focuses the motivation onto specifically sexual targets, increases copulatory rate and efficiency (5). The presence of testosterone is permissive for the dopamine (DA) release in the median preoptic area (MPOA), both during basal conditions and in response to a female. One means by which testosterone may increase dopamine (DA) release is by upregulating nitric oxide synthase (NOS), which produce nitric oxide (NO), which inturn increases dopamine (DA) release, which is ultimately facilitative to copulation (6).The ventromedian nucleus of hypothalamus (VMH) regarded as a center controlling female sexual behavior (7) Pfaff and Sakuma, (1979) which contains both nitric oxide synthase (NOS) (8, 9 , 10 , 11) and estrogen receptors (12).Estrogen may directly regulate the neuronal expression of nitric oxide synthase (nNOS) in the ventromedial nucleus and an increase in (nNOS) may result in elevated (NO) production and facilitation of lordosis behavior (13).The intro- cerebroventricular (ICV) microinjection of nitric oxide donor, facilitated lordosis in estrogen – primed rats in the absence of progesterone (14). It appears that progesterone normally causes the release of nitric oxide (NO) that stimulates (LHRH) secretion from the hypothalamus which facilitated lordosis in rats (14). The NO/cGMP pathway most likely mediates the facilitatory effects of α1- adrenoceptors on lordosis behavior in female rats and prior exposure of hypothalamus and (POA) to both estrogen and progesterone is required to link α1–adrenoceptors to this pathway. The adrenoceptor – NO- cGMP pathway, via activation of protein kinase G (PKG) is involved in the stimulation of female sexual behavior by vaginocervical stimulation (VCS) (15). The objective of this study was done to focus light on possible improving and enhancement of sexual activities of both male and female mice by using L-arginine as a precursor of nitric oxide (NO).
Materials and Methods
A total of (50) young adult male and (50) virgin female white albino mice, weighing 25-30 gm. with an average of 27.5 0.02 gm. were used. They were kept under suitable environmental conditions of 20-25oC. in an air conditioned room, (12) hours light and nourished adlibitum. Ten animals of each sex were given 200mg/kg B.W. of L-arginine orally daily for 15 and 30 days. Other groups of (10) mice of both sexes were likewise treated with 100 mg/kg B.W. of L-NAME and methylene blue 0.35mg/kg B.W. intraperitoneally (16). The study consists of three experiments; the first one is male aggressive behavior which used to test the aggressive behavior of treated male mice. In this test, a mouse was faced with a younger male in a neutral cage for (10) minutes and the parameters: Latency of aggressiveness (second), frequency of aggressiveness, and duration of aggressiveness (second) were recorded (17). The second experiment is male sexual behavior test which conducted by introducing a treatment male into the cage occupied by an estrous virgin female and the sexual activities of the treated male were observed directly for (3) hours. The parametersrecorded were: The latency of copulation (second), frequency of copulation (per-hour), and duration of copulation (second) (18), while the third experiment female sexual behavior test was tested in the male's home cage, using single housed male mice, after monitoring estrous cycle of each female mouse by taking vaginal smears. Each test lasted (3) hours and female responses to male mounts or intromissions were scored as receptive lordosis posture. The lordosis quotient(number of lordosis/number of mounts and intromissions) was recorded as female sexual behavior activity index (19). At the end of the experiment and evaluating of estrus phase of female mice, both male and female mice were anesthetized by diethylether, blood samples were obtained via cardiac puncture. Samples were centrifuged at (2500) rpm for (15) minutes and serum samples were stored in a freezer at (-18)0C till analyze to determine the hormonal levels of estrogen, progesterone, FSH, LH, for female mice and testosterone for male mice which were done by using commercial radio immunoassay (RIA) kits.
Data were analyzed by using completed randomized design in factorial experimental (Two- way) ANOVA. For calculation the effect of factors on dependent traits using (SPSS package 2008). All the data were analyzed by using the procedure of (20) and a probability of (P<0.05) was considered as significant differences.
Results and Discussion
The effects of L-arginine and its antagonists L-NAME (nitric oxide synthase inhibitior) and methylene blue (soluble guanylyl cyclase inhibitor) on the male aggressive behavior test parameters table (1) revealed a significant increase at (P<0.05) in latency of aggressiveness (second) a decrease in frequency of aggressiveness and a decrease in duration of aggressiveness (second) in L-arginine treated groups in both periods of treatment 15 and 30 days, while opposit results were observed in both of L-NAME and methylene blue treated groups in both periods of treatment as compared with control groups. The results of male sexual behavior test parameters table (2) showed a significant decrease at (P<0.05) in latency of copulation (second), increase in frequency of copulation (per – hour) and increase in duration of copulation (second) in both periods of treatment 15 and 30 days in L-arginine treated groups, while both of L-NAME and methylene blue treated groups showed abolishment of male sexual behavior parameters in both periods of treatment, while the effect of L-arginine on female sexual behavior parameter (lordosis quotient) table (3) revealed a significant increase at (P<0.05) in both periods of treatment 15 and 30 days as compared with control groups, and there were opposit results observed in both L-NAME and methylene blue treated groups which revealed abolishment in lordosis quotient in both periods of treatment 15 and 30 days. The results of testosterone hormonal levels table (4) showed a significant increase at (P<0.05) in hormonal levels in L-arginine treated groups and decrease in both L-NAME and methylene blue in both periods of treatment 15 and 30 days as compared with control groups, while there were significant increase at (P<0.05) in female hormonal levels of FSH, LH, estrogen and progesterone table (5) in L-arginine treated groups and decrease in these hormonal levels in both L-NAME and methylene blue treated groups in both periods of treatment 15 and 30 days as compared with control groups. The increase of aggressive behavior which observed in L-NAME and methylene blue treated groups in the present study might be attributed to the pharmacological inhibition of neuronal nitric oxide synthase (nNOS) by L-NAME by evokingelevatedaggression in male due to, reduction in serotonin (5-HT) turnover (21, 22, and 23). On otherwise, deficiencies in (nNOS) may result in increasing aggression through reducing serotonergic function in the hypothalamus and cerebral cortex and decreasing social investigation through decreasing in dopaminergic function, due to important role of nNOS –NO signaling which increases dopamine release in the median preoptic area (MPOA) of hypothalamus which, inturn increases sexual motivation and facilitates social memory(24 , 25 and 26). The increasing of male sexual behavior activitymight be attributed to the critical role of L-arginine – NO pathway in regulating dopamine release during copulation in the (MPOA) under the effect of testosterone which promote dopamine release in the (MPOA) of hypothalamus by upregulating nitric oxide synthase (NOS) activity in the (MPOA) (27,28 and 6).In addition, the highest sexual desire in male mice treated with L-arginine might be related to the increase of testosterone production from Leydig cells of testes (29 and 30) due to, the role of L-arginine – NO pathway in activatingthe hypothalamo – pituitary- gonadal axis and this triggers release of gonadoal hormones which ensure the rediness of the circuitry involved in copulation (31). The increase of female sexual behavior activity might be attributed to the increase of estrogen and progesterone hormonal levels by L-arginine – NO pathway which boost the effect of lordosis behavior and this result is in agreement with (32) which demonstrated that estradiol and progesterone are necessary for occurring sexual behavior and females must receive both estradiol and progesterone for displaying proceptive and be receptive to the males. In addition, normal vaginal cervical stimulation (VCS) of female mice by the male facilitates lordosis behavior and proceptivity through release of norepinephrine (NE) in the ventromedial hypothalamus (VMH) which inturn activates1– adrenergic receptors that affect lordosis behavior via NO-cGMP-PKG pathway (33, 34 and 35). Furthermore, nitric oxide (NO) acts in both brain and pituitary as a neuroendocrine regulator such as acts as a signal transducer in norepinephrine – induced prostaglandin E2 (PGE2) release from hypothalamic and both (NE) and (PGE2) regulate the pulsatile release of (GnRH/LH) surge which inturn regulate estrogen release from the ovary as well as enhancement of female sexual activity (36). Other explanation of increasing female mice hormonal levels is due to, effect of L-arginine – NO pathway on estrous cycle length after five periods of estrous cycles treatment which displayed a gradual increase in estrus phase length (16) beside its effect on intra and extra-follicular regulation in estrus phase which lead to increase hormonal levels of FSH/LH and estrogen in female mice. In conclusion, the present data show that L-arginine – NO pathway is more prominent in enhancement and improving the sexual desire and activities in both genders.
Table 1: Effects of treatment daily with L-arginine orally, L-NAME intraperitoneal and methyleneblue intraperitoneal on male mice aggressive behavior parameters.
Periods of treatment / 15 days / 30 daysLatency of aggressiveness second / Frequency of aggressiveness / Duration of aggressiveness second / Latency of aggressiveness second / Frequency of aggressiveness / Duration of aggressiveness second
D.W. as control 1 / 32.00±0.96Ab / 4.00±0.22Ab / 3.60±0.25Ab / 33.00±0.90Ab / 3.80±0.23Aba / 3.40±0.28Ab
Normal saline as control 2 / 33.00±0.90Ab / 3.40±0.02Ab / 4.20±0.20Ab / 32.20±0.96Ab / 3.00±0. 03Ab / 3.38±0.24Ab
L-arginine 200 mg/kg BW. / 40.40±0.20Ba / 1.20±0.04Aa / 1.90±0.09Aa / 46.20±0.22Ba / 1.00±0.05Aa / 1.24±0.03Aa
L-NAME 100 mg/Kg BW. / 6.80±0.28Ac / 24.41±0.07Ac / 41.60±1.04Ac / 5.00±0.30Bc / 28.00±0.01Bc / 45.00±0.31Bc
Methylene blue 0.35mg/Kg BW. / 7.00±0.26Ac / 23.20±0.14Ac / 41.80±1.02Ac / 4.80±0.35Bc / 27.66±0.05Bc / 45.30±0.33Bc
Values are presented as Mean ±SE
Small letters denoted to P<0.05 difference between treated groups.
Capital letters denoted to P<0.05 difference between periods of treatment.
Number = 5mice/group.
Table(2) : Effects of treatment daily with L-arginine orally, L-NAME intraperitoneally and methylene blue intraperitoneally on male sexual behavior parameters (sexual behavior test)
Periods of treatmentand parameters
Groups / 15 days / 30 days
Latency of copulation (second) / Frequency of copulation (per-hour) / Duration of copulation (second) / Latency of copulation (second) / Frequency of copulation (per-hour) / Duration of copulation (second)
D.W. as control 1 / 30.00±0.94Ab / 8.50±0.20Ab / 14.67±0.77Aa / 29.80±0.20Ab / 8.56±0.21Ab / 14.63±0.36Ab
Normal saline as control 2 / 30.00±0.94Ab / 8.50±0.20Ab / 14.60±0.77Aa / 29.60±0.20Ab / 8.50±0.21Ab / 14.63±0.36Ab
L-arginine (200) mg/kg B.W. / 12.00±0.32Ba / 18.40±0.74Ba / 29.94±1.90Bb / 8.20±0.37Aa / 20.0±0.54Aa / 33.00±0.56Aa
L-NAME (100) mg/Kg B.W. / - / - / - / - / - / -
Methylene blue (0.35) mg/Kg B.W. / - / - / - / - / - / -
Values are presented as Mean ±SE
Small letters denoted to P<0.05 difference between treated groups.
Capital letters denoted to P<0.05 difference between Periods of treatment.
Number = 5mice/group.
Table (3): the effect of treatment daily with L-arginine orally, L-NAME intraperitoneally and methylene blue intraperitoneally on female sexual behavior parameter (sexual behavior test).
Periods of treatmentand parameters
Groups / Lordosis quotient
15 days / 30 days
D.W. as control 1 / 0.39±0.05Ab / 0.40±0.07Ab
Normal saline as control 2 / 0.40±0.05Ab / 0.44±0.07Ab
L-arginine (200) mg / kg B.W. / 0.56±0.04Ba / 0.66±0.02Aa
L-NAME (100) mg / kg B.W. / - / -
Methylene blue (0.35)mg/Kg B.W. / - / -
Values are presented as Mean ±SE
Small letters denoted to P<0.05 difference between treated groups.
Capital letters denoted to P<0.05 difference between periods of treatment.
Number = 5mice/group.
Table (4): The testosterone hormonal levels (ng/ml) in male mice, L-arginine treated orally, L-NAME and methylene blue intraperitoneally
Periods of treatmentand hormonal
levels
Groups / 15 Days / 30 Days
D.W. as control 1 / 0.83+0.03Ab / 1.38+0.02Ab
Normal saline as control 2 / 0.80+0.04Ab / 1.42+0.01Ab
L-arginine (200) mg/kg B.W. / 1.93+0.02Ba / 23.38+0.01Aa
L-NAME (100) mg/kg B.W. / 0.55+0.01Ac / 0.28+0.03Ac
Mehylene blue (0.35) mg/kg B.W. / 0.50+0.04Ac / 0.30+0.02Ac
Values are presented as Mean + S.E.
Small letters denoted to significance P < 0.05 difference between treated groups.
Capital letters denoted to significance P < 0.05 difference between periods of treatment.
Number = 5 mice / group
Table (5): The hormonal levels (ng/ml) in female mice L-arginine treated orally, L-NAME intraperitoneally and methylene blue intraperitoneally daily
Periods oftreatmentand hormonal
levels
Groups / 15 Days / 30 Days
FSH / LH / Estrogen / Progesterone / FSH / LH / Estrogen / Progesterone
D.W. as control 1 / 4.98+0.70 Ab / 22.51+2.80 Ab / 10.70+0.59 Ab / 1.78+0.09 Aa / 5.08+0.37 Ab / 24.62+2.78 Ab / 11.11+1.94 Ab / 1.63+0.08 Aa
Normal saline as control 2 / 5.01+0.53 Ab / 21.53+2.69 Ab / 11.63+0.39 Ab / 1.85+0.09 Aa / 5.93+0.37 Ab / 22.68+1.14 Ab / 13.00+2.13 Ab / 2.03+0.38 Aa
L-arginine (200) mg/kg B.W. / 13.00+1.15 Ba / 49.80+1.11Ba / 24.80+2.61 Ba / 2.01+0.17 Ba / 16.70+2.04 Aa / 59.20+3.02 Aa / 29.00+1.97 Aa / 2.98+0.62 Ab
L-NAME (100) mg/kg B.W. / 1.98+0.31 Ac / 6.58+0.17 Ac / 2.34+0.20 Ac / 0.72+0.08 Ab / 1.05+0.08 Bc / 4.76+0.22 Ad / 2.01+0.36 Ac / 0.58+0.12 Ac
Methylene blue (0.35) mg/kg B.W. / 2.06+0.14 Ac / 7.03+0.14 Ac / 2.75+0.22 Ac / 0.89+0.03 Ab / 2.02+0.24 Ac / 8.81+1.45 Ac / 3.00+0.92 Ac / 0.95+0.08 Ac
Values are presented as Mean+SE
Small letters denoted to P < 0.05 difference between treated groups.
Capital letters denoted to P < 0.05 difference between periods of treatment
Number = 5 mice/group.
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