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The Protective Effect of Aromatase Inhibitor Anastrozole on Varicocele-Induced Testicular Dysfunction in Rats

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The Protective Effect of Aromatase Inhibitor Anastrozole on Varicocele-Induced Testicular Dysfunction in Rats Egyptian Journal of Basic and Clinical Pharmacology December 2017, Vol. 7, No. 2: 97-104 http://www.ejbcp.eg.net/ Original Article The Protective Effect of Aromatase Inhibitor Anastrozole on Varicocele-Induced Testicular Dysfunction in Rats Amany N. Ibrahim1, Magdy I. Attallah2, and Reham Abdelrahman Elnaggar3 1 Department of Clinical Pharmacology, Faculty of Medicine, Benha University 2 Department of Medical Pharmacology, Faculty of Medicine, kasr al-eini, Cairo University 3 Department of pharmacology and toxicology, Faculty of pharmacy, Misr University for science and technology (MUST) A B S T R A C T Copyright © 2017 Aim: This study aimed to investigate the protective effect of anastrozole on experimental varicocele - Amany N. Ibrahim induced testicular dysfunction in rats. et al. This is an open access article Material and method: adult male albino rats were divided randomly into three groups; group 1 act distributed under as control. In group 2 (varicocele rats) varicocele was induced by partial ligation of the left renal vein. the Creative Group 3 (anastrozole-treated group) varicocele rats received anastrozole (400mg/l) in the drinking water for a period of 9 weeks immediately after ligation of the left renal vein. Serum FSH, LH, Commons testosterone and intratesticular testosterone were measured. Seminal parameters (progressive motility, Attribution License, sperm cell concentration, epididymal sperm abnormal forms) were estimated. Testicular tissue which permits specimens were histopathologically examined by hematoxylin & eosin staining. unrestricted use, distribution, and Results :after 9 weeks of operation, the varicocele induced significant decreased of bilateral reproduction in any intratesticular testosterone levels and marked impairment of seminal parameters and histopathological medium, provided changes in the rat tests compared with control group (p<0.05). While, serum FSH, LH and serum the original work is testosterone levels insignificantly decreased. All these factors were significantly improved by anastrozole. These results clearly pointed the pivotal role of aromatase inhibitors in varicocele induced properly cited. testicular dysfunction. Key Words: Aromatase inhibitors, varicocele, testicular dysfunction, anastrozole. Corresponding Author: Amany N Ibrahim Email: [email protected] 1. INTRODUCTION Varicocele is observed in 10-20% of the general male Recent studies showed that aromatase inhibitors can population, in 35-40% of men with primary infertility increase endogenous testosterone production and serum and in up to 80% of men with secondary infertility testosterone levels. Treatment of infertile males with (Bechara et al., 2009; Razi et al., 2011). Varicoceles are the aromatase inhibitors testolactone, anastrazole, and being reported in up to 91% of sub-fertile cases, most of letrozole has been associated with increased sperm who were previously regarded as having idiopathic production and return of sperm to the ejaculate etiology (Resim et al., 1999). Recently, the pathogenesis in men with non-obstructive azoospermia (Tadros and mechanisms by which varicocele induce testicular Sabanegh, 2017; ShoshanySchlegel, 2017).Aromatase degeneration, spermatogenesis arrest and finally is a cytochrome p-450 enzyme that converts testosterone infertility are not completely understood. The suggested to estradiol and androstenedione to estrone. Aromatase mechanisms include reflux of toxic metabolites from found in the testis, liver and brain in addition to female adrenal and renal origin, impairment of the reproductive tract and adipose tissue. In the testes, hypothalamic-gonadal axis, venous stasis leading to aromatase are localized to Leydig and Sertoli cells and testicular hypoxia and elevation of temperature in are found in germ cell tumors (Inkster et al., 1995). testicles (Comhaire, 1991; Benoff and Gilbert 2001). Thus, the aim of the present study was to evaluate the The previous studies indicated that the intra-testicular possible protective effects of anastrozole-aromatase testosterone decreases in varicocele patients (Naughton inhibitor- on the testicular dysfunction through et al., 2001). measurement of the serum level of testosterone, LH and FSH, evaluation of seminal parameters and 97 The Protective Effect of Aromatase Inhibitor Anastrozole on Varicocele-Induced Testicular dysfunction in Rats histopathological changes in experimentally-induced Serum level of FSH: varicocele in rats. The serum FSH, was measured with the 2. MATERIALS AND METHODS commercially available kits, follicle stimulating hormone (Amer-sham pharmacia, Biotech Ltd., Buckinghamshire, 2.1. Experimental protocol U.K.). 2.1.1 Drugs and chemicals: Serum levels of LH: were measured by using Anastrozole: AstraZeneca UK Ltd. lutenizing hormone assay system (Amersham Pharmacia, Biotech Ltd., Buckinghamshirc, U.K.). Urethane crystal: (Ethyl carbamate). (Prolabo, Paris). Serum levels of testosterone: Diagnostic Products Corporation. Los Angeles. U.S.A). 2.1.2 Animals used 2.2.2 Intratesticular testosterone: Fifty adult male albino rats, weighting (120-150gm). They were brought from Experimental Animal Breeding At the end of the experiment the rats were sacrificed Farm, Helwan - Cairo .All animals were housed in and testes were cut and weighed. Right testes controlled laboratory condition at 20 -25C in a 12h homogenized in 10 ml distilled water and undergo light/dark cycle and had free access to standard centrifugation at 3000 rpm for 10 min. at 4 ºC the laboratory chow (El-Nasr Company, Abou-Zaabal, supernatal fraction was used as a sample to determine Cairo, Egypt) and water. They have acclimatized for one testicular testosterone. week and were caged (10/cage) in fully ventilated room 2.2.3 Seminal parameters: at room temperature. All experimental protocols were approved by the ethics committee. The epididymal content of each rat was obtained after cutting the tail of epididymis and squeezing it gently in 2.1.3 Experimental design: sterile clean watch glass and examined using the Sixty rats were randomly divided into three groups: following parameters: Group (1): Control group, a suture was placed Progressive motility: around the renal artery but not tied. This group did not The progressive motility of sperms was receive any drug. examined according to the method reported by Bearden Group (2): varicocele non- treated rats. Rats were and Fluquary (1980), where a small droplet of semen anaesthetized with urethane (white crystals 0.6ml/100g was added to one drop of sodium citrate solution 2.9% on 25% freshly prepared solution). Varicocele was done by a warm slide. Several fields were examined and the a clamp passed behind the left renal vein distally to the incidences of progressively motile sperms were spermatic vein insertion. A silk ligature was placed estimated and recorded. around the left renal vein at this site and was tied over the Sperm cell concentration: top of a probe. The probe was then withdrawn and the vein was allowed to expand (Turner, 2001). This was performed according to the technique adopted by Bearden and Fluquary (1980), using pipette Group (3): varicocele treated: rats were treated with of haemocytometer. The undiluted semen was withdrawn anastrozole (400mg/L) in the drinking water for 9 weeks up to the mark 0.1 and the pipette was then filled up to (Turner et al, 2000). the mark 101 by normal saline stained with eosin. The 2.2.1 Parameters used: contents of the pipette were mixed by holding the end of the pipette with thumb and index fingers and shaking it 2.2.1 Hormonal assay parameters: vigorously. The cover slide was placed over the counting Serum levels of FSH, LH, and testosterone: chamber and a drop of diluted semen was spread between the haemocytometer slide and its cover. The sperms in 5 At the end of the 9th week rats were anaesthetized large squares contains (contain 80 small squares) were with urethane. Blood samples were collected from the counted using the high power objective lens. The sperm abdominal aorta to determine the serum levels of FSH, cell concentration in cubic milliliters was estimated by LH and testosterone hormone. The blood samples multiplying the counted number of sperms by 10 (depth) (2mleach) were allowed to clot at room temperature, and 1000 (dilution). centrifuged at 3000 rotation/minute and the sera were separated. Samples were stored at -20 Ć in dark Epididymal sperm abnormal forms: containers. A drop from epididymal content of each rat was mixed with an equal drop of eosin-nigrosin stain. The semen was carefully mixed with the stain. Then films were spread on clean slides. A hundred sperms were 98 Amany N. Ibrahim et al. examined at random per slide under high power objective 3.3 Seminal parameters: lens and percentage of abnormal sperms was recorded. 3.3.1 Sperm count changes: 2.3. 1 Histopathological techniques: In varicocele non- treated rats, there was significant The testes were removed and half of each testis was decrease in sperm count p<0.05 compared to control fixed in Bouin’s solution (Bouin, 1897), and embedded group. Administration of anastrozole (400mg/L) in paraffin wax sectioned at 5 microns (Harris, 1900) significantly increase sperm count, compared with and stained with haematoxillin and eosin stain. varicocele non- treated group but it was at non- Histological changes were observed by light microscopy. significant lower level (p<0.05) if compared to control group. Statistical Analysis: was performed using one-way analysis of variance (ANOVA) to detect
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