1. The Role of Nitric Oxide in Testicular Sperm Extraction (TESE) Canan Hurdag 1, Yasemin Ersoy Canıllıoglu 2, Asli Kandil 3, Meral Yuksel 4, Evrim Unsal 1, Vildan Karpuz 5. 1 Department of Histology and Embryology, Medical Faculty, Istanbul Bilim University, 2 Department of Histology and Embryology, Medical Faculty, Bahcesehir University, 3 Department of Biology, Science Faculty, Istanbul University, 4 Vocational School of Health Related Professions, Marmara University, 5 Department of Pathology, Medical Faculty, Istanbul Bilim University, Istanbul-Turkey eNOS reactions were intensely observed in primer spermatocytes, Sertoli cells, miyoid cells and Leydig cells of spermatozoa detected groups (Figure 1A) compared to spermatozoa absent groups (Figure 2A). eNOS reaction in spermatozoa detected group was considerably higher than spermatozoa absent group (Graphic 1) which shows that eNOS plays an important role in spermatogenesis detection. iNOS reaction were dense in Sertoli cells, miyoid cells and Leydig cells in spermatozoa absent groups (Figure 2B) compared to spermatozoa detected groups (Figure 1B). iNOS reaction was also higher in comparison to eNOS reaction. It was observed that iNOS reaction was higher than spermatozoa absent groups (Graphic 2). nNOS reactions were showed Leydig cells in the interstitial area both spermatozoa detected groups (Figure 1C) and spermatozoa absent groups (Figure 2C). There was no significant difference in the nNOS reaction between the spermatozoa detected and spermatozoa absent groups (Graphic 3). NO release of sperm detected group was 111,9 ± 65,5 rlu/mg tissue and in spermatozoa absent group 29,8 ± 18,5 rlu/mg tissue. There was a significantly higher NO level in spermatozoa detected group (p<0,0001) (Graphic 4). Superoxide radical generation in spermatozoa detected groups were significantly lower than spermatozoa absent groups (10,3 ± 2,3 rlu/mg tissue vs; 19,4 ± 7,2 rlu/mg tissue; p=0,0003) (Graphic 5). Peroxynitrite ratio in spermatozoa absent group was significantly higher than spermatozoa detected group. Peroxynitrite ratio in spermatozoa absent group (42,5 ± 17,8 %) was significantly higher than spermatozoa detected group (19,1 ± 11,5 %; p=0,0038) (Graphic 6). INTRODUCTION MATERIAL-METHOD RESULTS Many conditions or events associated with male infertility are inducers of oxidative stress. Such stress conditions can cause changes in the dynamics of testicular microvascular blood flow, endocrine signaling, and germ cell apoptosis. Testicular oxidative stress appears to be a common feature in much of what underlies male infertility. In recent years, scientists have studied the role of nitric oxide (NO) in the male infertility. There are different physiological roles for NO in male reproductive system. NO is a potent vasodilator and cell signaling molecule can play its own role in amplifying testicular injury. The aim of study is to evaluate NO and oxidative stress in testicular tissue in infertile men with azoospermia cases either do not have spermatozoa or have any spermatozoa. In this study, testicular biopsies were obtained from 20 men with azoospermia who were attended to infertility center for diagnosis or infertility treatment. TESE samples were divided to two groups as spermatozoa were detected and not detected for azoospermic men. Immunohistochemistry was used to localize the all three of nitric oxide synthase (NOS) isoforms (endotheial NOS (eNOS), inducible NOS (iNOS), neuronal NOS (nNOS)) in these tissues. At the immunohistochemical procedures, tissues were incubated eNOS rabbit polyclonal primer antibody (1:50) for eNOS, iNOS rabbit polyclonal primer antibody (1:100) for iNOS and nNOS mouse monoclonal primer antibody (1:25) for nNOS reactions. Chemiluminescence measurement of NO is based on the reaction of hydrogen peroxide and NO to peroxynitrite. Chemiluminescence measurements were made at room temperature using a luminometer (Junior LB 9509, EG & G Berthold, Bad Wildbad, Germany). Specimens were divided into two pieces and put into vials containing 2 mL of PBS+HEPES buffer (0,5 M phosphate buffered saline containing 0,02 M HEPES; pH 7,4). Superoxide radical were quantitated after the addition lucigenin enhancer for a final concentration of 0.2 mM (1,3). Chemiluminescence measurement of NO is based on the reaction of hydrogen peroxide and NO to peroxynitrite. In this system K2CO3 (0.4 mM), desferrioxamine (60 mM), H2O2 (4 mM) ve luminol-sodium salt (3.6 mM) was added to the tube containing the testis specimen and 2 mL of PBS+HEPES buffer (2, 4)( Kikuchi K, Nagano T, Hayakawa H, Hirata Y, Hirobe M: Detection of nitric oxide production from a perfused organ by a luminol-H2O2 system. Anal Chem 65:1794–1799, 1993.. However peroxynitrite also may form in testes by a reaction between superoxide and NO. To differentiate these two sources NO levels were measured after addition of 0.5 mM carboxy-PTIO, a NO scavenger. The difference between the measurments indicated the level of peroxynitrite forming in testes tissues. Counts were obtained at 1 minute intervals for a counting period of 5 minutes. Results were given as the area under curve (AUC) of relative light unite and corrected for wet tissue weight (AUC of rlu/mg tissue) CONCLUSION These results showed that three isoforms of eNOS and iNOS play an important role in spermatogenesis process in azoospermic men. nNOS may also act as a signal molecule for spermatogenesis process. Biochemical testing indicated that NO formation in spermatozoa absent patients were higher than spermatozoa detected patients. Our results showed an increase in NO formation via chemiluminescence, measurement of eNOS and iNOS increased causing NO generation. At the same time iNOS mediated NO generation to react with superoxide radical and produced peroxynitrite. In biological systems superoxide and nitric oxide can react and produce peroxynitrite radical, which is very toxic to cell membranes and other cellular components. In this study, peroxynitrite ratio in spermatozoa absent patients was more than two-fold than of spermatozoa detected patients. Superoxide dismutase (SOD) activity, which is an enzyme that scavenges superoxide and converts it to hydrogen peroxide, in higher measured in pachytene spermatocytes, round spermatids, and spermatozoa than Sertoli cells. In conclusion that different categories of testicular cells display variable susceptibility to oxidative stress, and also superoxide radical generation. The study demonstrated that superoxide radical generation was significantly higher in spermatozoa absent patients testis tissues, than from spermatozoa detected patients testes. In conclusion, testicular oxidative stress plays a significant role in male infertility. Figure 1 A-C: At the testis tissue of the sperm detected group, A) eNOS reaction at the seminifer tubule, primer spermatocyte (  ), Sertoli cells (  ), miyoid cells (  ), the cells in the tubule lumen ( ,  ), Leydig cells (  ) and blood vessels (BV), B) iNOS reaction at the seminifer tubule, spermatogonium (  ), primer spermatocyte (  ), Sertoli cells (  ), miyoid cell (  ), the cells in the tubule lumen(  ) and Leydig cells (  ), C) nNOS reaction at the Leydig cells in the interstitial area (  ). Figure 2 A-C: At the testis tissue of the sperm absent group, A) eNOS reaction at the seminifer tubule, Sertoli cells (  ), miyoid cells (  ), blood vessels (BV) and Leydig cells (  ), B) iNOS reaction at the seminifer tubule, Sertoli cells (  ) and Leydig cells (  ), C) nNOS reaction at the Leydig cells in the intertitial area (  ). Graphic 1: Distribution of the eNOS immunreaction at the testis tissue of the assay groups. ***p<0.0001. Graphic 2: Distribution of the iNOS reaction at the testis tissue of the assay groups. *p<0.04. Graphic 3: Distribution of the nNOS reaction at the testis tissue of the assay groups. BV A A B B C C Graphic 4: Nitric oxide concentrations of spermatozoa detected groups and spermatozoa absent groups. p<0.0001. Graphic 5: Superoxide generation of spermatozoa detected groups and spermatozoa absent groups. p=0.0003. Graphic 6: Peroxynitrite ratio of spermatozoa detected groups and spermatozoa absent groups. p=0.0038.