The Role of Nitric Oxide on Spermatogenesis in Infertile Men with Azoospermia
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D J Med Sci 2021;7(1):7-19 doi: 10.5606/fng.btd.2021.25040 Original Article The role of nitric oxide on spermatogenesis in infertile men with azoospermia Canan Hürdağ1, Yasemin Ersoy Çanıllıoğlu2, Aslı Kandil3, Meral Yüksel4, Ayşe Altun5, Evrim Ünsal6 1Department of Histology and Embryology, Medical Faculty of Demiroğlu Bilim University, Istanbul, Turkey 2Department of Histology and Embryology, School of Medicine, Bahçeşehir University, Istanbul, Turkey 3Department of Biology, Science Faculty, Istanbul University, Istanbul, Turkey 4Vocational School of Health Related Professions, Marmara University, Istanbul, Turkey 5Department of Obstetrics and Gynaecology School of Medicine, Istanbul University, Istanbul, Turkey 6Genart Woman Health and Reproductive Biotechnology Center, Ankara, Turkey ABSTRACT Objectives: The underlying pathophysiological mechanisms of azoospermia is still unclear. The aim of the study was to evaluate nitric oxide synthase (NOS) isoforms and free radical release in testicular sperm extraction (TESE) in infertile men with azoospermia. Materials and methods: The study included 40 men (mean age: 37.2±2 years; range 25 to 55 years) with azoospermia which were divided into two groups: spermatozoa-present (n=20) and spermatozoa-absent (n=20). Testicular samples were examined morphologically, immunohistochemically, and biochemically. The TESE samples were examined according to number of mast cells stained with toluidine blue; immunohistochemically with three types of NOS isoforms, and free radicals were measured with chemiluminescence method, respectively. Results: Endothelial NOS (eNOS) reaction in spermatozoa-present group was considerably higher than spermatozoa-absent group (p<0.001). Compared to the spermatozoa-present group, inducible NOS (iNOS) reaction was higher than the spermatozoa-absent group (p<0.05). Neuronal NOS (nNOS) reaction was only prominent in Leydig cells in both groups. Mast cells increased (p<0.05) in the interstitial area surrounding seminiferous tubules in spermatozoa-absent samples. Superoxide radical generation in spermatozoa-present samples was significantly lower (p=0.0003). The peroxynitrite ratio in spermatozoa-absent samples was significantly higher (p=0.0038). Conclusion: These results suggest that eNOS, iNOS, and mast cells play an important role in spermatogenesis process in azoospermic men. Keywords: Male infertility, mast cell, nitric oxide, oxidative stress, testicular sperm extraction. Fertile men are defined as males whose partner rates.[3] Sperm retrieval (SR) is based on the conceived within 12 months after stopping use type of azoospermia which is either obstructive of contraception.[1] Male fertility requires the (OA) or non-obstructive (NOA). Testicular sperm production of a sufficient number of mature extraction (TESE) is the technique of choice for and motile spermatozoa. A defect in any of NOA.[2] these parameters may cause male infertility. Azoospermia is the absence of spermatozoa in the Several studies have shown that high levels ejaculate. Surgical methods have been developed of reactive oxygen species (ROS) are related to retrieve spermatozoa from the epididymides and with male infertility.[4-6] Excessive amounts of the testes of patients.[2] After sperm acquisition, ROS produced by leukocytes and immature intracytoplasmic sperm injection (ICSI) can spermatozoa can cause damage to the normal be applied which results in higher fertilization spermatozoa.[6-8] Situations in which ROS are Received: January 18, 2021 Accepted: January 27, 2021 Published online: May 05, 2021 Correspondence: Ayşe Altun, MD. İstanbul Üniversitesi Tıp Fakültesi, Kadın Hastalıkları ve Doğum Anabilim Dalı, 34093 Fatih, İstanbul, Türkiye. Tel: +90 551 - 389 66 47 e-mail: [email protected] Cite this article as: Hürdağ C, Ersoy Çanıllıoğlu Y, Kandil A, Yüksel M, Altun A, Ünsal E. The role of nitric oxide on spermatogenesis in infertile men with azoospermia. D J Med Sci 2021;7(1):7-19. 8 D J Med Sci associated also involve mast cell activation.[9] paraffin. The sections were, then, stained with Nitric oxide (NO) is one of the ROS which regulates the Masson’s trichrome to examine connective the physiology of the reproductive function. It is tissue and stained with Toluidine blue to define synthesized by nitric oxide synthase (NOS) which mast cells, and NOS isoforms and were examined exists in three known forms: endothelial NOS immunohistochemically, while the seminiferous (eNOS), inducible NOS (iNOS), and neuronal tubules were graded according to the Johnson NOS (nNOS).[10] score.[11] In the present study, we aimed to evaluate Masson’s trichrome staining protocol for NOS isoforms and free radical release in TESE in connective tissue infertile men with azoospermia. The slides were deparaffinized, immersed in graded alcohol and finally rinsed in water. MATERIALS AND METHODS The slides were, then, fixed with the Bouin’s TESE and sperm analysis fixative. Then, each slide was immersed and left in Weigert’s iron hematoxylin solution In this prospective cohort study, ejaculate for approximately 10 min. Each slide was samples were taken from male patients who passed through acid fuchsin, acetic acid, applied for in vitro fertilization (IVF) and phosphomolybdic acid, and Aniline blue solution, were evaluated according to the 2010 World respectively. Finally, the slides were rinsed in Health Organization (WHO) sperm analysis 2% acetic acid and washed with distilled water. criteria[1] between Februrary 2011 and June Once the slides were dehydration with Clear® 2013. The samples were taken twice from each xylene, they were ready to be examined under patient with sexual abstinence between two microscopy.[12] and seven days. The specimens were examined and analyzed with a Makler® sperm counting Toluidine blue staining protocol for chamber (Sefi Medical Instruments Ltd., Haifa, mast cells Israel). When there was no sperm in the The sections were deparaffinized and pellet of the centrifuged sample, male patients hydrated with distilled water. The slides were, were referred for TESE operation. Testicular then, immersed in toluidine blue. After staining, biopsy was taken and a specimen size of the sections were passed through distilled 2 to 3 mm was obtained, where the seminiferous water, alcohol, and xylene respectively over tubules were opaque and dilated which were a period of time. For mast cell counting, five later examined morphologically. The study fields were selected from each slide at ¥200 protocol was approved by the Istanbul Bilim magnifications and the obtained values were University, Medical Faculty, Ethics Committee evaluated statistically.[11] (2011/SAG-022). The study was conducted in accordance with the Declaration of Helsinki. Immunohistochemical analysis Histological and immunohistochemical The sections (3 µm) were deparaffinized with analysis xylene and rehydrated with ethanol and finally with water. Antigen retrieval was accomplished A total of 40 male patients (mean age: 37.2±2 in the Decloaking Chamber™ in a citrate buffer years; range 25 to 55 years) were included (pH 6.0). The endogenous peroxidase activity for persistent azoospermia. The study did not was blocked with 3% H O . Blocking reagent take into account the patient’s medical history, 2 2 was applied to each slide, followed by 5-min physical examination, or hormonal profile. incubation at room temperature in a humid Testicular biopsy samples in infertile men with chamber. Sections were incubated for overnight azoospermia were divided into two groups as at 4°C with rabbit polyclonal iNOS antibody, follows: spermatozoa-present [sperm (+) group] eNOS antibody, and nNOS. The sections were (n=20) and spermatozoa-absent [sperm (-) group] biotinylated goat anti-rabbit antibodies. Slides (n=20). were washed in PBS after which streptavidin Testicular samples were fixed in the Bouin’s peroxidase label reagent was applied. The colored solution. The samples were embedded in product was incubated in 3, 3’-diaminobenzidine, The role of nitric oxide on spermatogenesis in infertile men with azoospermia 9 tetrahydrochloride, dihydrate (DAB). The slides between peroxynitrite and NO levels, the were counterstained with Mayer’s hematoxylin. measurement was done from the same test The intensity and the distribution of NOS tube after addition of 0.5 mM carboxy- staining were scored by histological (H-score) PTIO-potassium salt (2-(4-carboxyphenyl)- value. 4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt), a NO scavenger. The difference Determination of free radicals between the measurements indicated the level For the determination of the superoxide of peroxynitrite forming in testicular samples. anion, NO, and peroxynitrite radicals in All counts were obtained at 1-min intervals for testicular samples of azoospermia, we used a total period of 5 min. The results were given the chemiluminescence (CL) method. The as the area under the curve (AUC) of relative CL measurements were made by using a light unite and corrected for wet tissue weight luminometer. The TESE sample was divided (AUC of rlu/mg tissue). into two and put into vials containing 2 mL of PBS+HEPES buffer (0.5 M PBS containing Statistical analysis 0.02 M 4-(2-hydroxyethyl), piperazine- Statistical analysis was performed using the 1-ethanesulfonic acid; pH 7.4). Superoxide GraphPad Software Inc., CA, USA. Descriptive radicals were quantitated after the addition of data were expressed in mean ± standard lucigenin enhancer for a final concentration of deviation (SD),