Conclusions Nitric oxide has vigor on Polycystic Ovarian Syndrome (PCOS) 1. Dept of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran 2. Dept of Pathology, School of Medicine, Shahed University, Tehran, Iran 3. Dept of Embryology, Royan Institute for Reproductive Biomedicine Research Center, Tehran, Iran MethodsObjectivesResults To explore the involvement of nitric oxide (NO) in Polycystic Ovarian Syndrome (PCOS) (Fig. 1.) through over- activation of NO synthase enzyme (NOS) the present work was designed. The enzyme activation was investigated using NADPH- diaphorase technique. Animals were female Wistar rats (weighing g) housed in standard conditions. Animals were tested for taking their vaginal smears. The stage of estrous for each animal was evaluated using an adaptation of the papanicolaou (PAP) stain used for humans as developed by Dr. George Papanicolaou [1]. The animals showing diestrous phase were then grouped into the experimental and control categories. The experimental groups were intra- perituneally injected L-arginine ( mg/kg) through a period ranging 9 to 14 days/once a day. Control groups only received saline (1 ml/kg, ip) through the all experiments. At the end of the work animals were got out by diethyl-ether overdose and the ovaries were immediately excised from the rats and trimmed from periovarian fat and bursa. The ovaries were immersed in phosphate buffered saline (PBS, pH: 7.0) for hr at 4 ºC, followed by cryoprotection in 30% sucrose buffer. Sections (5-9 μm thick) were cut on a cryostat and mounted onto poly-l-lysine coated slides. The slides were washed with Triton X-100 while being shacked (2 min). The slides then were incubated with 1 mg/ml NADPH, 0.2 mg/ml NBT (Nitro blue tetra zolium) at 37 °C overnight. This histochemical reaction is known as a marker for all isoforms of NOS [2]. Ovarian morphology In experimental groups, ovarian cysts were formed at a significant level. The cysts in L-arginine- exposed rats resembled those described by Green and Glodzieher [3] in human PCO cysts. NADPH- diaphorase reactivity Positive NADPH-d reactivity was observed in L- arginine treated rats’ ovaries (Fig. 2B). but no reactivity was shown in control groups (Fig. 2A). PCOS is now categorized in the inflammatory phenomena. NO is considered as a pro- inflammatory agent due which the cystic formations may appear in ovaries of the treated animals. This research may propose a force of NO in PCOS. Fig. 2. A-B, Survey views showing control, L-arginine-exposed rat ovaries in the same magnification. A, Ovary from normal control rat showing cumulus oophorus. B, Cystic ovary from a L-arginine- exposed rat with highly positive reaction to NADPH. control L-Arginine 1)Hbscher CH, Brooks DL, Johnson JR. A quantitative method for assessing stages of the rat estrous cycle. Biotechnic and histochemistry. 2005; 80(2): ) Dawson TTD, Bredt DS, Fotuhi M. Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc Natl Acad Sci. USA. 1991; 88: )Green JA, Glodzieher JW. The Polycystic Ovary. IV. Light and electron microscope studies. Am J Obstet Gynecol. 1965; 91: )Hbscher CH, Brooks DL, Johnson JR. A quantitative method for assessing stages of the rat estrous cycle. Biotechnic and histochemistry. 2005; 80(2): ) Dawson TTD, Bredt DS, Fotuhi M. Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc Natl Acad Sci. USA. 1991; 88: )Green JA, Glodzieher JW. The Polycystic Ovary. IV. Light and electron microscope studies. Am J Obstet Gynecol. 1965; 91: Fig.1. Pathophysiological characteristics of PCOS.