1. The Effects of NOS Isoforms In Human Normospermia, Asthenospermia And Oligospermia Cases H. Karakaya 1, E. Ünsal 1, A. Kandil 2, Y. E. Canıllıoğlu 1, C. Hürdağ 1 1 Department of Histology and Embryology, Medical Faculty, Istanbul Bilim University, Istanbul-Turkey 2 Department of Biology, Science Faculty, Istanbul University, Istanbul-Turkey canan.hurdag@istanbulbilim.edu.tr canan.hurdag@istanbulbilim.edu.tr Male factor infertility accounts for up to half of all case of infertility and affects 1 in 20 men in the general population. Evidence now suggests that reactive oxygen species (ROS) mediated damages to sperm is a significant contributing pathology in 30-80% of all cases (1). The highly biologically active free radical is nitric oxide (NO) (2). NO is known to play important functional roles in a variety of physiological system as well as in the male reproductive system. In this study we aimed to examine both inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) isoforms by using immunohistochemistry methods in human normospermia, asthenospermia and oligospermia groups and also determined nitrite/nitrate concentrations in the seminal plasma. INTRODUCTION Three groups of 20 volunteers were selected with proven fertility with normospermia semen profiles (sperm number ≥20X10 6 /ml, progressive motility ≥50%, and normal morphology ≥30%), oligospermia (sperm number≤20X10 6 /ml, progressive motility ≥50%, normal morphology ≥30%) and asthenospermia (sperm number ≥20X10 6 /ml, progressive motility ≤50%, normal morphology ≥30%). The samples were taken from donors were the same age (Graph 1). Samples were treated with collection fluid. Then they were centrifuged and pellets were prepared for sandwich model by using a cyto-block kit. Samples were fixed in 4% formalin and embedded in paraffin. The paraffin sections were applied by immunohistochemistry method. Sections were incubated for overnight at 4°C with rabbit polyclonal iNOS antibody (iNOS Ab-1, Rabbit PAb, RB-1605-P, NeoMarkers Fremont, CA) and eNOS antibody (eNOS Rabbit PAb, RB-1711-P Neomarker Fremont, CA). Nitrite/nitrate concentrate in seminal plasma was determined in these groups and statistical analysis of the data were performed using GraphPad Prism version 5.0 for Windows (GraphPad Software, USA). MATERIAL-METHOD eNOS reactions were observed in the equatorial region, partial membrane of the head and mid-piece of normospermia samples (Figure 1a). It was observed that eNOS reactions decreased in all region in the cross sections taken from asthenospermia and oligospermia samples compared to the normospermia groups (Figure 1b-c). iNOS reactions were dense in the acrosomal region of normospermia donors (Figure 2a). iNOS reactions showed moderate intensity in head membrane and equatorial region in the cross sections obtained from asthenospermia groups compare to the normospermia groups. However the staining was highly intense in the mid-piece region and the acrosome (Figure 2b). The staining of oligospermia sections with iNOS showed that acrosome region of the sperm was intensely stained while the partial membrane of the head and the neck region of the sperm was less intensely stained (Figure 2c). Nitrite/nitrate concentrations increased in seminal plasma of asthenospermia (p<0,05) and oligospermia (p<0,05) donors compared to the normospermia groups (Graph 2). RESULTS Many researches have stated that NO has a role in the spermatozoa functions (3). eNOS is known to be present in physiologic functions of human spermatozoa (3). It was detected that eNOS reactions were dense in normospermia samples; however, it decreased in asthenospermia and oligospermia compare to normospermia groups. However, It was seen that iNOS reactions were both different in localization and density in asthenospermia and oligospermia samples when compared to normospermia. In asthenospermia groups, iNOS reaction was caused intensive staining in the mid-piece region of immotile spermatozoa where mitochondria is located which led us to believe that the damaged mitochondria can be the cause of immobility of spermatozoa. Our immunohistochemistry results confirm NO is a key role in sperm functions. Physiological and pathological processes depend on the relative level of nitric oxide and oxidative stress. The increased of nitrite/nitrate in seminal plasma leads to decreased in sperm motility and viability. CONCLUSION Graph 1: Age of normospermia, asthenospermia and oligospermia donors. Graph 2: Nitrite/nitrate concentrations in seminal plasma of normospermia, asthenospermia and oligospermia donors. Figure 1a-c: eNOS reactions in the equatorial region (  ), partial membrane of the head (  ) and mid-piece (  ) of sperms. a) Normospermia, b) Asthenospermia, c) Oligospermia samples. Figure 2a-c: iNOS reactions in acrosome (  ), the equatorial region (  ), partial membrane of the head (  ) and mid-piece (  ) of sperm. a) Normospermia, b) Asthenospermia, c) Oligospermia samples. References: 1-Tremellen K. Oxidative Stress and Male Infertility-a Clinical Perspective. Human Reproduction Update 2008.14 3:243-258. 2- Aitken J.R. 2006. Reactive Oxygen Species:Friend or foe. In: Jonge C. and Barratt C,editor. Sperm Cambridge University Press: Edinburgh, UK.,pp170-194. 3- M. B. Herrero and C. Gagnon. Nitric oxide: a novel mediator of sperm function. Journal of Andrology, Vol. 22, No. 3, May/June 2001. a a a a b b c c b b c c