DEPARTMENT OF ZOOLOGYBANARAS HINDU UNIVERSITYVaranasi-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

ACKNOWLEDGEMENTIt 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 OFLITERATURE

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 Ca2+ and Calmodulin as coactivators and are accelerated by tetrahydrobiopterin

NOR

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 asribonucleotide reductaseandaconitase, activation of the solubleguanylatecyclase, ADP ribosylation of proteins, protein sulfhydryl groupnitrosylation, and iron regulatory factor activation. NO has been demonstrated to activateNF-Bin 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 Ca2+and the opening of calcium-activated potassium channels. The fall in concentration of Ca2+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 O2. 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(BH4) 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 Ca2+-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 Ca2+ 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 Ki values of 15 nM, 39 nM, and 4.4 M for nNOS (bovine), eNOS (human), and iNOS (mouse), respectively. The reported Ki value for the inhibition of iNOS ranges from 4-65 M. L-NAME inhibits cGMP formation in endothelial cells with an IC50 of 3.1 M (in the presence of 30 M arginine) and reverses the vasodilation effects of acetylcholine in rat aorta rings with an EC50 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-NAMEMetabolism 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 25c 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. H2SO4. 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% H2SO4 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 standardAscorbic 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 H20) 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 KNO3.

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 (H2O2) 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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