DEPARTMENT OF ZOOLOGY

BANARAS HINDU UNIVERSITY

Varanasi-221005


This is to certify that Ms. Komal Jaiswal (Enrolment number: 345083) has completed the
dissertation work for the fulfillment of the requirements for the degree of Master of Science
(M.Sc.) of Banaras Hindu University. The dissertation work, entitled Effect of NOS inhibitor
drug (L-NAME) on reproduction of female laboratory mouse (Mus musculus) embodies
the results of her investigations conducted during the period she worked as a M. Sc. student. I
recommend the dissertation to be submitted for evaluation for the award of the degree of Master
of Science of Banaras Hindu University.



Prof. ( Mrs.) C. M. Chaturvedi Head
Supervisor Department of Zoology








ACKNOWLEDGEMENT
It is my pleasure to express my profound sense of gratitude to Prof. (Mrs.) Chandra Mohini
Chaturvedi, Department of Zoology , Banaras Hindu University , not only for suggesting
problem for my M.Sc. dissertation but also for her valuable guidance, constant
encouragement and moral support throughout the period of my dissertation work.
I am thankful to the Head of the Department Prof. C.M.Chaturvedi for providing
Departmental facilities.
I wish to express my special regards to Prof. Chandana Haldar, Prof. Amitabh Krishna,
Prof. Shio Kumar Singh, Prof. B. Lal and Prof. Pranab lal Pakrashi for their valuable teaching
and creating interest in reproductive physiology which helped my dissertation work.
I express my sincere thanks to my seniors Mr.Vineet Prakash Singh ,Ms.SunitaYadav, Ms.
Saba Shahin , Mr. Somanshu Banerjee , Mr. Arun Kr Yadawa , Ms. Rashmi Richa, Mr. Manoj
Kumar Jha and Ms. Pallavi Srivastava for their help and encouragement during my dissertation
days.
I owe my thanks to laboratory attendants Mr. Raj Kumar Singh and Mr. Birju for their help in
various forms.
I am very much thankful to Ms. Dipanshu Joshi for their kind help. I want to thank my friends
Kavita, Shilpa, Kuvardeepu, Jitendra, Sandipan, Anirban, Jayita, Aradhana, Arun, Khushboo and
Paromita for their continuous curiosity about the topic of my dissertation work, discussion and
suggestion of new plans which made me able to do my work properly.
It is difficult to find suitable words to express my deep sense of gratitude for the inspiration,
keen interest, love and encouragement; I received during the period from my parents, Mr. Sanjay
Jaiswal and Mrs. Kiran Jaiswalfor always supporting and believing in me.


Date: Komal Jaiswal




CONTENT

INTRODUCTION
REVIEW OF LITERATURE
MATERIALS & METHODS
RESULTS
DISCUSSION
REFERENCES





















INTRODUCTION














INTRODUCTION:-
In the late 1970s, Dr. Robert Furchgott observed that acetylcholine released a substance that
produced vascular relaxation, but only when the endothelium was intact. Furchgott and Zawadski
(1980) provided evidence that acetylcholine-induced relaxation of vascular rings was mediated
by a non-prostanoid factor. Initially, Furchgott called this substance endothelium-derived
relaxing factor (EDRF), but by the mid-1980 he and others identified this substance as being NO
(Palmer et al. 1987and Ignarro et al. 1987). This observation opened a new field of research and
eventually led to his receiving a Nobel prize. Garthwaite et al. (1988) also provided evidence for
the presence and synthesis of NO in brain. During the period from 1988 to 1992, research on the
biology and functions of NO escalated, and then in 1992, NO was named Molecule of the
Year by D.E.Koshland, (Jr., Editor for Science. In view of the significant role of this molecule
in body physiology, specially reproduction this project is focused on NO inhibitor on female
reproduction or laboratory mouse.








REVIEW
OF
LITERATURE






NITRIC OXIDE (NO):-
Nitric oxide or nitrogen oxide, also known as nitrogen monoxide, is a molecule with chemical
formula NO. It is a free radical in mammals including humans. Nitric oxide (NO), is a highly
reactive, diffusible and unstable radical, plays an important role in the regulation of a wide range
of physiological processes, including cellular immunity, angiogenesis, neurotransmission, and
platelet aggregation.NO is an important cellular signalling molecule. It may also function as a
retrograde neurotransmitter. NO synthesis is carried out by nitric oxide synthase (NOS), which
comes in three different forms: neuronal nitric oxide synthase (nNOS), inducible nitric oxide
synthase (iNOS) and endothelial nitric oxide synthase (eNOS). These different isoforms share
a similar NO synthesis reaction mechanism, which uses L-arginine as a substrate and O2 and
NADPH as co-substrates to form NO (Palmer et al.1988).Marletta et al. (1988)reported that
macrophages generate nitrite and nitrate from L-arginine. Free NO is a transient species with a
half-life of only about five seconds. Hence, most studies on NO action are based on the activity
of NOS. Reaction of NO with O2 in aqueous solutions produces relatively unreactive nitrate and
nitrite ions as products. However, NO can rapidly react with superoxide to produce highly
reactive peroxynitrite (ONOO-). Almost all biological effects of NO are achieved either directly
or through other reactive nitrogen intermediates.Arginine-derived NO synthesis has been
identified in vertebrates including mammals, invertebrates and bacteria. Best studied are
mammals, where three distinct genes encode NOS isozymes: Neuronal (nNOS or NOS-1),
Cytokine-inducible (iNOS or NOS-2) and Endothelial (eNOS or NOS-3).nNOS and iNOS are
soluble and found predominantly in the cytosol, while eNOS is membrane associated. Evidence
has been found for NO signaling in plants, but plant genomes are devoid of homologs to the
superfamily which generates NO in other kingdoms.
TYPE OF NOSs:-There are mainly three type of NOSs are present.
Name Gene(s) Location Function
Neuronal NOS
(nNOS)
NOS1 nervous tissue
skeletal muscle type II
cell communication
Inducible NOS
(iNOS)
NOS2 immune system
cardiovascular system
immune defence
against pathogens
Endothelial NOS
(eNOS)
NOS3 endothelium vasodilation
SYNTHESIS OF NO:-
Nitric oxide is formed from L-arginine by the action of nitric oxide synthase (NOS). The L-
arginine is converted to NO in two successive steps, Inthe first step two electron oxidation of L-
arginine occurs and it is converted into N-w-hydroxy-L-arginine. In the second step, this N-w-
hydroxy-L-arginine is converted into nitric oxide and L-citruline, utilizing one and half NADPH
and O2. Both step require Ca
2+
and Calmodulin as coactivators and are accelerated by
tetrahydrobiopterin



R
NO
BIOLOGICAL FUNCTIONS OF NITRIC OXIDE:-
Nitric oxide is now known to play important functional roles in a variety of physiological
systems. Within the vasculature, NO induces vasodilation, inhibits platelet aggregation, prevents
neutrophil/platelet adhesion to endothelial cells, inhibits smooth muscle cell proliferation and
migration, regulates programmed cell death (apoptosis) and maintains endothelial cell barrier
function. NO generated by neurons acts as a neurotransmitter, whereas NO generated by
macrophages in response to invading microbes acts as an antimicrobial agent. Because neurons,
blood vessels and cells of the immune system are integral parts of the reproductive organs. NO is
an important regulator of the biology and physiology of the reproductive system. Indeed, in the
past 10 years, NO has established itself as a polyvalent molecule which plays a decisive role in
regulating multiple functions within the female as well as the male reproductive system. TheNO
play an important role in various reproductive organs under physiological and pathological
conditions.
ROLE OF NO IN REPRODUCTION:-
Nitric oxide (NO) plays a crucial role in reproduction at every level in the organism. In the brain,
it activates the release of luteinizing hormone-releasing hormone (LHRH). The axons of the
LHRH neurons project to the mating centers in the brain stem and by afferent pathways evoke
the lordosis reflex in female rats. In males, there is activation of NOergic terminals that release
NO in the corpora cavernosa penis to induce erection by generation of cyclic guanosine
monophosphate (cGMP). NO also activates the release of LHRH which reaches the pituitary and
activates the release of gonadotropins by activating neural NO synthase (nNOS) in the pituitary
gland. In the gonad, NO plays an important role in inducing ovulation and in causing luteolysis,
whereas in the reproductive tract, it relaxes uterine muscle via cGMP and constricts it via
prostaglandins (PG) It is already apparent that nitric oxide plays a crucial role in reproduction at
all levels from the brain to the gonads and to the accessory sex organs.
ROLE OF NO IN MATING BEHAVIOR:-
LHRH involved in mediating male sex behavior. Studies in vivo have shown that NO stimulates
the release of LHRH that induces sex behavior. This behavior can be stimulated by 3V injection
of SNP and is blocked by inhibitors of NOS. Apparently, there are two LHRH neuronal systems:
one, with axons terminating on the hypophyseal portal vessels, the other with axons terminating
on neurons which mediate sex behavior. NO is also involved in inducing penile erection by the
release of NO from NOergic neurons innervating the corpora cavernosa of the penis. The role of
NO in sex behavior in both sexes has led us to change the name of NO to the sexual gas.

ROLE OF NO IN ACCESSORY SEX ORGANS:-
As indicated earlier, NO activates LHRH neurons projecting to the brain stem. These brain stem
neurons activate NOergic neurons in the pelvic plexus that innervate the accessory sex organs,
including the penis in males and the vagina in females.
In the penis, these terminals release NO that activates soluble guanylatecyclase in the smooth
muscle of the corpora cavernosa, generating cGMP that causes relaxation of penile smooth
muscle as shown by Ignarro (Burnett AL et al. 1992). This allows erection to occur as blood
enters via the penile arteries. Indeed, nitroglycerin applied to the surface of the penis or SNP
injected into the penis will induce erection in man and rat (McCann SM, unpublished data);
however, the NO produced reaches the general circulation and can produce undesirable side
effects such as headache and decline in blood pressure.These side effects have been
circumvented to a degree by the development of Viagra, a phosphodiesterase inhibitor that more
or less selectively inhibits the phosphodiesterase found in the corpora cavernosa of the penis.
A comparable organ in the female is the vagina that has a weak smooth muscle constrictor at its
external orifice. The vaginal constrictor relaxes and the vaginal mucosa secretes vaginal fluid on
sexual arousal in women. NO secreted from the NOergic pelvic neuronal terminals in the vaginal
wall may cause vaginal relaxation and secretion via liberation of cGMP. Indeed, arousal in
women also appears to be enhanced by Viagra probably by delaying the breakdown of the cGMP
produced.
MECHANISM OF ACTION OF NO:-
There are several mechanisms by which NO has been demonstrated to affect the biology of
living cells. These include oxidation of iron-containing proteins such as ribonucleotide
reductase and aconitase, activation of the soluble guanylatecyclase, ADP ribosylation of proteins,
protein sulfhydryl group nitrosylation, and iron regulatory factor activation. NO has been
demonstrated to activate NF-B in peripheral blood mononuclear cells, an important
transcription factor in iNOS gene expression in response to inflammation.
It was found that NO acts through the stimulation of the soluble guanylatecyclase, which is a
heterodimeric enzyme with subsequent formation of cyclic-GMP. Cyclic-GMP activatesprotein
kinase G, which causes reuptake of Ca
2+
and the opening of calcium-activated potassium
channels. The fall in concentration of Ca
2+
ensures that the myosin light-chain kinase (MLCK)
can no longer phosphorylate the myosin molecule, thereby stopping the crossbridge cycle and
leading to relaxation of the smooth muscle cell.



Fig.1 Schematic diagram of the role of NO in transcellular signal transduction. NOS increases its activity in
response to intracellular Ca2+ influx, which stimulates, via calmodulin (CaM), the NOS enzyme. NOS
catalyses the conversion of O2 and L-arginine to NO and L-citrulline. Activation of NOS requires
nicotinamide adenine dinucleotide phosphate (NADPH) as cofactor. NO diffuses to No responsive target cell
where it binds to a heme moiety of soluble guanylylcyclase (sGC) which, following activation, catalyses cyclic
GMP (cGMP) formation. Possible NO target may be heme moiety of another hemoprotein, mainly,
cyclooxygenase (COX) which, following activation, converts arachidonic acid (AA) into prostaglandin E2
(PGE2). Thus, PGE2 activates adenylatecyclase causing an increase in cAMP. Since both cGMP and cAMP
are second messengers, they can affect multiple enzymatic pathways in target neurons.

NITRIC OXIDE SYNTHASE (NOS):-
Nitric oxide synthases (NOSs) are a family of enzymescatalyzing the production of nitric oxide
(NO) from L-arginine. Different members of the NOS family are encoded by separate
genes.NOS is one of the most regulated enzymes in biology. There are three known isoforms,
nNOS , eNOS are constitutive (cNOS) and the third is inducible (iNOS). NOSs are unusual in
that they require fivecofactors. Eukaryotic NOS isozymes are catalytically self-sufficient. The
electron flow in the NO synthase reaction is: NADPH FAD FMN heme O
2
.
Tetrahydrobiopterin provides an additional electron during the catalytic cycle which is replaced
during turnover. NOS is the only known enzyme that binds flavin adenine dinucleotide (FAD),
flavin mononucleotide (FMN), heme, tetrahydrobiopterin(BH
4
) and calmodulin.
STRUCTURE OF NOS:-

Fig.2 Structure of Nitric oxide synthase (NOS). (Berridge M.J.2012)
These enzymes exist as homodimers, each monomer consisting of two major domains: an N-
terminal oxygenase domain and a C-terminal reductase domain. The interdomain linker contains
the calmodulin-binding sequence. These three isoforms exhibit similarities in their structure and
mechanism of action. Calmodulin is required for the activity of all three isoforms. The activation
of the constitutively expressed isoforms requires Ca
2+
-dependent binding of calmodulin to the
enzyme. However, in the case of iNOS, calmodulin is irreversibly bound to the enzyme and its
activity is regulated by its rate of synthesis rather than by Ca
2+
concentration. The reductase
domain contains the FAD and FMN moieties. The oxygenase domain, which contains the
binding sites for heme, tetrahydrobiopterin, and arginine. The maximal rate of NO synthesis is
established by the intrinsic maximum ability of the reductase domain to deliver electrons to the
heme domain.
NITRIC OXIDE SYNTHASE (NOS) INHIBITORS:-
NOSs are inhibited by a wide variety of substances, e.g. L-arginine analogues, haem binding
imidazole andindazole derivatives, calmodulin antagonists, redoxactive dyes, and flavoprotein
inhibitors (Fukuto & Chaudhuri, 1995). Of all these potential NOS inhibitors, Ng-derivatives of
the substrate L-arginine have proven most useful for specific inhibition of NO biosynthesis in
pharmacological experiments and clinical trials. Ng-methyl-L-arginine (L-NMMA) was used to
demonstrate the precursor role of L-arginine in NO formation by activated macrophages (Hibbs
et al., 1987) and vascular endothelial cells (Palmer et al., 1988). L-NMMA was shown to
attenuate endothelium-dependent relaxations both in vivo and in vitro (Rees et al., 1989a, b) and
to block NO synthesis by endothelial cell homogenates (Mayer et al., 1989; Palmer & Moncada,
1989). Since then, many more L-arginine based NOS inhibitors have been described (Fukuto &
Chaudhuri, 1995). NG-nitro-L-arginine (L-NOARG) and its methyl ester (L-NAME) were
identified as potent inhibitors of endothelial and neuronal NO synthesis (Mulsch & Busse, 1990;
Rees et al., 1990; Moore et al., 1991).
L-NAME (hydrochloride):-

Fig. 3. Chemical structure of L-NAME(L-NG-Nitroargininemethyl ester)

L-NAME requires hydrolysis of the methyl ester by cellular esterases to become a fully
functional inhibitor (L-NNA). L-NNA exhibits some selectivity for inhibition of neuronal and
endothelial isoforms. It exhibits K
i
values of 15 nM, 39 nM, and 4.4 M for nNOS (bovine),
eNOS (human), and iNOS (mouse), respectively. The reported K
i
value for the inhibition of
iNOS ranges from 4-65 M. L-NAME inhibits cGMP formation in endothelial cells with an IC
50

of 3.1 M (in the presence of 30 M arginine) and reverses the vasodilation effects of
acetylcholine in rat aorta rings with an EC
50
of 0.54 M.
It is found that the apparent inhibitory potency of L-NAME solutions was closely correlated to
the rates of its hydrolysis to the free acid, indicating that L-NAME is an inactive prodrug of the
active inhibitor, L-NOARG. The arginine analogues L-NAME and L-NOARG have been shown,
in vitro and in vivo, to be potent inhibitors of NOS.

Fig. 4. Mechanism of action of L-NAME
Metabolism of l-name is mainly catalyzed by blood cell esterases. The very rapid metabolism of
L-NAME in perfused hearts (l/2 time-2 min) suggests a high esterase activity in the coronary
endothelium, agreeing with the generally high metabolic capacity of vascular endothelial cells
Gerritsen, (1987). Brouillet et al. (1995) have recently shown that L-NAME is rapidly
metabolized in vivo to L-NOARG and the potent neurotoxin methanol, suggesting that L-NAME
metabolism may produce detrimental effects unrelated to NOS inhibition.





















MATERIALS
&
METHOD






ANIMALS:-
Laboratory mice, Mus musculus (order: Rodentia ; family : Muridae) were used and they were
obtained from the mice colony of central animal house , Institute of Medical Science (IMS) and
maintained in our laboratory mice room. Mice were maintained under hygienic conditions in
well ventilated room at 25

c under photoperiod regimen of LD 12:12.All mice were kept in

polypropylene cages with dry rice husk as the bedding material. Animals were supplied with
food (standard rodent food pellets supplied by Pashu Aahar Kendra; Varanasi) and tap water ad
libitum.
NOS INHIBITOR DRUG L-NAME:-
Full name: -N-Nitro-L-arginine methyl ester hydrochloride,
Molecular Formula: -C7H15N5O4 HCl,
Molecular Weight: -269, 69 g/mol,
Product Number: - N5751,
Brand name: - Sigma,
CAS-No.:-51298-62-5,
Company: -Sigma-Aldrich Chemicals Pvt Limited Plot No 12 Bommasandra - Jigani Link Road
560100 BANGALORE INDIA.
EXPERIMENTAL PROTOCOL:-
Adult (3 weeks) female laboratory mice of weighing 15-25 gram were used in the investigation.
The female mice were randomly selected and divided into three groups each group containing
four animals.
CHECKING OF OESTROUS CYCLE BEFORE TREATMENT:-
Before the starting of drug treatment, mice were weighed individually and vaginal smear of all
the mice was checked continuously for 5 days. All the mice showed normal cyclicity.
Group I (control):- Mice of the control group receives normal saline daily (0.1ml).
Group II, Low dose L-NAME (LD):-Mice of this group receives low dose of L-NAME (1mg
/100 gm body weight).
Group III, High doseL-NAME (HD):-Mice of this group receives high dose of L-NAME (2mg
/100 gm body weight).
All the injections (normal saline as well as L-NAME) were given intrapetonially (i.p.)
At the end of the experiment, final body weight was recorded and animals were sacrificed by
decapitation after 24 hours of last injection.Just after the decapitation blood of each mouse was
collected and centrifuged (4000 rpm for 20 minutes) to collect plasma which was stored in -20c
for total Nitric oxide assay and ELISA of estrogen. Ovary and uterus were dissected out,
weighed and fixed in PFA/ Bouins fluid for histology. Part of this tissue were also processed for
biochemical analysis.
Whole experiment was conducted in accordance with Institutional practice and within the
framework of the revised animals (scientific procedures) act of 2002 of the Govt. of India on
animal welfare.


PARAMETERS:-
Following parameters were monitored in the present study-
Body weight
Reproductive cyclicity (estrous cycle)
Weight of reproductive tissue
Ovarian Ascorbic acid assay
Nitrate nitrite estimation
ELISA of plasma estradiol
Counting of number of follicles and corpus luteum
Histology of ovary and uterus

ESTRUS CYCLE:-
Before starting the treatment vaginal smear of all the mice was checked for 5 days. Which
showed normal cyclicity. Cyclicity was again checked during last 5 days of treatment period.
OVARIAN ASCORBIC ACID ESTIMATION:-
Ovarian ascorbic acid was estimated by Schaffert & Kingsley method.(1955)described as
follows-
Principle: -WhenL-ascorbic acid was treated with TCA/DCIP it forms dihydroascorbate which
form diketoglucoronic acid after reaction with conc. H
2
SO
4
. This diketoglucoronic acid form
osazone by reaction with DNPH, which produce brown colour. Intensity of colour was measured
at 512 nm.
Procedure :-10% homogenate of each ovary was made and centrifuged at 3000rpm for 5 min.
Supernatant was taken in test tubes and 4% TCA (trichloroaceticacid) was added to make it 4ml.
1drop thiourea and 0.1ml DCIP (Dichloroindophenol) were then added and mixed well. After
adding 1ml of DNPH (Dinitrophenylhydrazine) test tubes were incubated at boiling water bath
for 10min. Then all the test tubes were placed at crushed ice and ~ 5ml of 85% H
2
SO
4
was added
slowly. After waiting for 10min OD was taken at 515 nm. The amount of ascorbic acid was
expressed in mg/g of tissue after calculating as follows-
Calculations:-
OD of ovary 1OD of standard
Ascorbic acid concentration (mg/g) = ---------------------------------------
Weight of ovary (g)

TOTAL NITRITE AND NITRATE ESTIMATION:-
NO is a reactive free radical and it is generally oxidized in NOx (nitrite/ nitrate). NO is a short
lives molecule but nitrite and nitrate are stable breakdown products of NO. Total nitrite and
nitrate concentration were measured in plasma and gonads by method of Sastry et al. (2002)
Briefly, for plasma nitrite and nitrate concentration, blood was collected in a heparinised
tube and centrifuged at 3000xg for 30 min to separate plasma. 10 % tissue homogenate was
prepared in 0.01 M phosphate buffer pH 7.4. To 100 l of each sample (plasma and gonads/
hypothalamus homogenate) or standard was added 400 l of carbonate buffer followed by a
small amount (~0.15 mg) of activated copper-cadmium alloy filings and incubated at room
temperature with thorough shaking. At the time of use, the alloy was washed with carbonate
buffer and dried on a filter paper. The reaction was stopped by the addition of 100 l of 0.35M
NaOH followed by 120 Mm ZnSO4 solution under vortex and allowed to stand for 10 min.
Tubes were then centrifuged at 8000xg for 10 min. 100 l of aliquots of clear supernatant were
transferred into the wells of a microplate (in quadriplicate) and Griess reagent (50l of
1% sulphanilamide prepared in 2.5% orthophosphoric acid and 50l of 0.1% N-
naphthylethylenediamine prepared in distilled H
2
0) was added to it. After 10 min, the
absorbance was read at 545 nm in an ELISA reader (ECIL, India). A standard graph was plotted
against different concentrations (0, 20, 40, 60, 80 and 100 M) of KNO
3
.

HORMONE ASSAY (ELISA OF17- ESTRADIOL):-
Serum level of estradiol was measured to evaluate the steroidogenic activity of the ovarian
follicle. Estradiol level was measured by using 17- estradiol ELISA kit (Diametra, REF
DKO003; Lot 2130).
Principle: -It is a competitive type of ELISA. The principle behind this assay was that estradiol
(antigen) in the sample competes with horseradish peroxidase estradiol (enzyme-labeled antigen)
for binding onto the limited number of anti- estradiol (antibody) sites on the microplates wells
(solid phase). After incubation, then on specifically bound antigen separation is performed by a
simple solid phase washing. The colour intensity is inversely proportional to the estradiol
concentration in the sample.
Procedure: - For standard 25l of standard estradiol solution was given in well and in rest of
the well 25 l blood serum was loaded. Then 200l of conjugate was added. After that this
reaction mixture was kept for 2hrs incubation at 37C. After incubation this reaction mixture was
removed and washed the each well 3-4 times with 300l wash solution. Then 100l enzyme
substrate (H
2
O
2
) and the TMB-substrate (TMB) were added in each well. After an appropriate
time (30 min in dark) has elapsed for maximum colour development, the enzyme reaction was
stopped by adding 100l stop solution in each well and the absorbencies are determined at
450nm. Estradiol concentration in the sample is calculated based on a series of standard.

HISTOLOGY:-
For histology, ovary and uterus were fixed in freshly prepared paraformaldehyde solution (PFA),
dehydrated in graded ethanol series, cleared in xylene, and embedded in paraffin. Tissues were
sectioned at 7m, and sections were stained with periodic haematoxylin and counter stained with
eosin. The stained sections were examined under a light microscope.
Identification of different types of follicles as well as corpus luteum in mouse ovary was
performed. Changes in the histology of ovary and uterus were observed under microscope.
COUNTING OF FOLLICLES AND CORPUS LUTEUM NUMBER:-
Follicles and corpus luteum number was simply counted under binocular light microscope.
Ovary was taken in a cavity block with saline. First bursa (a thin covering present on ovary) was
removed by forceps and then all the follicles and corpus luteum was separated and counted.
STATISTICAL ANALYSIS:-
All data were analysed by one way analysis of variance (ANOVA), followed by post hoc Dunnet
test for comparison of groups from control. Difference was considered significant at P < 0.05
against control group.










RESULTS













DISCUSSION









Body weight of mice was altered by the administration ofonly high dose of NOS inhibitor drug
but the mice of all the groups appear to be healthy without any adverse effect. A significant
increase in the ovarian weight of treated mice occurred due to accumulation of stromal and non-
follicular deposits within the ovary as well as increased amount of fat deposition around the
ovary. However in contrast to this, the number of follicles decreased in L-NAME treated mice
because this NOS inhibitor also inhibits the folliculogenesis. It also inhibits proliferation of
thecal cells and granulosa cells of ovarian follicles. Number of developing follicle was decreased
possibly because of less steroidogenic activity.L-NAME is capable of inhibiting the NO
production and NO is essential for folliculogenesis and ovulation. Hence possibly in the
experimental group significant increase in atretic follicle number were observed. This suggestion
gets strengthen by the supporting parameters indicating decreased steroidogenesis, for eg.
number of atreatic follicles increased suggesting regressive change in ovarian physiology. In
experimental group low level of estradiol may be due to presence of less number of developing
follicles. L-NAME inhibits the steroidogenesis so the rate of follicular development became low
and ultimately the estradiol level falls in L-NAME treated groups. The level of ovarian ascorbic
acid which is inversely related to steroidogenesis, increased significantly in treated mice supports
L-NAME induced suppression of steroidogenesis. Above all a significant decrease in the
concentration of plasma estradiol of high dose of L-NAME treated mice further supports the
gonadosuppressive effect of NOS inhibitor in female mice.
L-NAME is a NOS inhibitor drug that inhibits the NO production via inhibiting the cGMP
pathway. So gradual decrease in NO level was observed in experimental groups. Decreased NO
level in treated mice indicates that this NOS inhibitor drug induced its NO suppression. Further
the present study clearly indicates that suppressed NO activity has induced ovarian suppression
exhibiting parallel relation of NO with ovarian activity. Increased size of uterus is attributed to
hyperplasia andswelling of endometrium of HD treated mice may be possible due to drug
induced inflammation like situation prior to implantation and the action of some critical
cytokines as well as prostaglandins which needs to be confirmed. However, physiologically
decreased uterine endometrial thickness clearly indicates suppression of ovarian function which
in turn inhibits the activity of accessory sex organ, the uterus in the present study,
The present study clearly supports parallel relation between NO and ovarian activity. Since L-
NAME suppresses NO activity which in turns decrease in ovarian function it is suggested thatthe
optimum level of NO is required for normal ovarian function, folliculogenesis and plasma
estradiol level.
However at this point it is worths mentioning the opposite relation in male mice. (Singh and
Chaturvedi 2013, 2014). In case of testicular function, inverse relation has been reported in NO
gonadal / testicular activity not only in control but in experimental condition. In sexual immature
and mature male mice, the amount of plasma and testicular NO activity was higher and lower
respectively indicating opposite relation between NO activity and testicular function. This
contrast in female (parallel relation) and male (inverse relation) mice in terms of NO-gonadal
relation although an interesting observation its underlying basis needs to be investigated.
Although sexual dimorphism in many aspects of body physiology is not surprising including
different age of puberty attainment in two sexes and different thyroid gonad relationship.
Thus NO is essential for ovarian function and maturation. Present findings clearly indicate that
inhibition of NO level can interrupt the ovarian activity (steroidogenesis, follicular maturation
and oocyte development) showing the important role of NO in female reproduction. Any
elevation or declination of NO level from its optimum level may lead to affect the ovarian
activity adversely.






















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