EFFECTS OF RECOMBINANT HUMAN INTERFERON-α-2b ON TESTICULAR MORPHOLOGY, TESTOSTERONE PRODUCTION AND AROMATASE GENE IN ALBINO RAT MODEL A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Anatomy By Salman Ahmed Farsi Kazi MBBS,M.Phil Department of Anatomy Faculty of Medicine & Allied Medical Sciences Isra University, Hyderabad, Sindh October 2016 EFFECTS OF RECOMBINANT HUMAN INTERFERON-α-2b ON TESTICULAR MORPHOLOGY, TESTOSTERONE PRODUCTION AND AROMATASE GENE IN ALBINO RAT MODEL By Salman Ahmed Farsi Kazi MBBS,M.Phil Name Supervisor/Co-supervisors Prof. Dr. M. Ghiasuddin Shah Rashdi Ph.D Prof. Dr Fatehuddin Khand Ph.D Dr Jameel Ahmed Gandahi Ph.D DEDICATION This thesis is dedicated to my beloved mother and father My wife, kids and my teachers CERTIFICATE This is to certify that Dr Salman Ahmed Farsi Kazi s/o Muhammad Khan Kazi has carried out research work on the topic “Effects of Recombinant Human Interferon-α-2b on testicular morphology, testosterone production and aromatase gene in albino rat model” under my supervision and that his work is original and his thesis is worthy of presentation to Isra University for awarding the degree of “Doctor of Philosophy” in the subject of Anatomy. Prof. Dr. M. Ghiasuddin Shah Rashdi Supervisor _____________________ Signature of Supervisor iv ACKNOWLEDGEMENT With the deep and profound sense of gratitude and thanks to the almighty ALLAH for giving me the chance for completing this thesis, I am greatly indebted to my respected Supervisor, Professor Dr Ghiasuddin Shah Rashdi and Co-supervisors Professor Dr Fatehuddin Khand and Dr Jameel Ahmed Gandahi for their cooperation, guidance and constructive criticism in the successful completion of this thesis and without their help, this manuscript was not possible to complete. I am grateful to Prof. Dr. Ghulam Qadir Kazi, the Vice Chancellor Isra University for his whole heartedly valuable co-operation and support. My thanks are also to all my colleagues, friends and well wishers and special thanks to Professor Dr A.G. Arijo, Professor Dr Shankar Lal Rathi, Prof. Dr Muhammad Bachal Bhutto, Prof. Dr Ahmed Tunio and Prof. Dr Aqeel Memon for giving me the support and help during the course of conduct of this study. v ABSTRACT BACKGROUND: Interferons (IFNs) are a family of biologically active natural proteins, secreted by the immune system. IFNs are produced by immune cells in response to viral, bacterial infections and cancers. The effects of IFN-α on testicular morphology and functions have never been clearly elucidated. Animal studies have shown conflicting results. OBJECTIVE/S OF STUDY: The objectives of this study were: 1. To investigate the effects of recombinant human interferon-α-2b (rh-INF-α-2b) on morphology of testes in adult albino rat model. 2. To observe the effects of rh-INF-α-2b on sperm morphology. 3. To determine the effects of rh-INF-α-2b on testicular hormone production. 4. To examine the effects of rh-INF-α-2b on hypothalamo-pituitary-testicular axis 5. To study the effect on rh-INF-α-2b on the aromatase gene. SUBJECTS AND METHODS: The present Experimental study was carried out at Animal house, Department of Animal Husbandry and Veterinary Sciences Sindh Agriculture University Tando Jam, Isra University Hyderabad and Diagnostic & Research Laboratory, LUMHS Hyderabad/Jamshoro over 3 year duration. Eighty adult rats (N=80) were selected by non-probability sampling and divided into; Group I: Control group receiving normal (0.9%) saline injections (n=20), Group II: Injections of recombinant human interferon-α-2b (3MIU) (n=20), Group III: Injections of recombinant human interferon-α-2b (5MIU) (n=20), Group IV: Injections of recombinant human interferon-α-2b (10MIU) (n=20). Albino rats were selected in a systemic way according to inclusion and exclusion criteria. The research proposal was submitted to ethical review committee (ERC) of Isra University. ERC approval vi also was taken from the animal ethical committee from the Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, Tando Jam. Animals were housed in stainless steel cages (with saw dust bedding) at optimal standard conditions of light and temperature. The recombinant human interferon-α- 2b was administered as intra-peritoneal injections thrice a week for three weeks. Animals were left for one week more. At 30th day of post-interferon injections, all rats were given anesthesia. Blood samples were collected by cardiac puncture and sera were used for hormonal assay. Sperm were isolated by 20 gauge needle attached disposable syringe from Epididymis. Smears were prepared by Leishman`s, Hematoxylin & Eosin stains for microscopy. Tissue slides thus obtained were observed and studied under microscope. Macro & Microscopic photography were carried out. Testosterone, Luteinizing and Follicle stimulating hormones (LH & FSH) were detected by ELISA method. Aromatase Gene Detection was performed by RNA extraction and RT-PCR using published primers. Data were analyzed using SPSS version 21.0 (Chicago, Illinois, USA) for windows release. RESULTS: Serum testosterone reduced progressively in interferon treated animals at different doses compared to control. Serum testosterone, mean± SD, in control groups was 0.81± 0.04 versus 0.74±0.03, 0.67±0.04 and 0.55±0.6 pg/mL in groups II, III and IV, respectively (p≤ 0.011). Serum FSH reduced progressively in interferon treated animals at different doses compared to control. Serum FSH, mean± SD, in control groups was 7.43± 0.79 compared to 6.6±0.75, 6.02±0.82 and 5.2±1.18 mIU/ml, respectively (p≤ 0.026). Serum LH also showed a reduction in interferon treated animals at different doses compared to control. Serum LH, mean± SD, in control groups was 7.56± 0.70 compared to 6.8±0.63, 5.65±0.92 and 4.7±1.31 vii mIU/ml, respectively (p≤ 0.003). Thickness of seminiferous tubules in control, rhINF- 3MIU, rhINF-5MIU and rhINF-10MIU was noted as 195.55±5.40, 181.20±7.82, 169.20± 2.35 and 155.90± 7.08 µm, respectively. Diameter of seminiferous tubules in control, rhINF-3MIU, rhINF-5MIU and rhINF-10MIU was noted as 9.37±2.09, 14.21±3.17, 16.97± 3.79 and 15.22± 3.40 µm, respectively. Seminiferous tubule length was reduced in experimental rats compared to control. Length of seminiferous tubules in control, rhINF-3MIU, rhINF-5MIU and rhINF-10MIU was noted as 13.52± 0.81, 12.86± 0.80, 12.08± 0.62 and 11.28± 0.62 µm, respectively. Germ cell counts were reduced in experimental rats compared to control. Germ cell counts in control, rhINF-3MIU, rhINF-5MIU and rhINF-10MIU were reduced in 2, 11, 17 and 19 rats, respectively. Germ cell maturation was arrested in experimental rats compared to control. Germ cell maturation arrest in control, rhINF-3MIU, rhINF-5MIU and rhINF- 10MIU was noted in 0, 03, 16 and 19 rats, respectively. Thick BM was found in more numbers of experimental rats compared to control. Increased thickness of BM was prominent in the high dose rhIFN-10MIU treated rats. Interstitial edema was noticeably noted in the experimental rats compared to control. Interstitial edema was noticeable finding in high dose rhIFN-10MIU treated rats. Hypervascularity was a prominent feature of the rhIFN treated rats on histopathological examination. Increased Sertoli cell counts were noted in the rhIFN treated experimental rats. Increased Sertoli cell counts in control, rhINF-3MIU, rhINF-5MIU and rhINF-10MIU were noted in 0, 13, 16 and 18 rats, respectively. Increased Leydig cell counts were noted in the rhIFN treated experimental rats compared to control. Increased Leydig cell counts in controls, rhINF-3MIU, rhINF-5MIU and rhINF-10MIU were noted in 0, 12, 16 and 19 rats, respectively. Texture studies of controls revealed that the viii seminiferous tubules were normal looking with intact basement membranes, epithelial cells layers, Sertoli cells and interstitial cells of Leydig. Sperm morphology of controls, rhIFN 3 MIU and 5 MIU showed abnormal morphology. Rats treated with rhIFN 3 MIU, rhIFN 5 MIU and rhIFN 10 MIU showed abnormalities. Low sperm counts, mixed premature and mature sperms, short tail, abnormal head & tail, plasma cells and double tails were observed in interferon treated experimental rats. Aromatase gene showed changes in INF treated rats. CONCLUSION: Our results suggest that rh-INF-α-2b disturbs the anatomy and physiology of testes. Interferons reduce the serum testosterone, serum luteinizing and follicle stimulating hormones through hypothalamic-pituitary-testicular axis (HPT) and also through direct inhibitory effects on the testicles. Interferons disturb the phenotype of gross anatomical and histological features of testes. Aromatase gene showed changes in INF treated rats. In conclusion, data herein demonstrated that 3, 5 and 10 mIU of recombinant human IFN-α2b exerted marked impact on sperm quality and fertility capacity of male rats. KEYWORDS: Interferon Aromatase gene Testosterone Follicle stimulating hormone Luteinizing hormone Histopathology ix LIST OF ABBREVIATION ABBREVIATION TERM AchE Acetylcholine esterase ACV Apple cider vinegar virus AE Aromatase enzyme AEG Aromatase enzyme gene AIDS Acquired immuno deficiency syndrome ANOVA Analysis of variance Anti HRP Horse reddish peroxidase APC Antigen presenting cell ATP Adenosine tri phosphate BM Basement membrane BSA Body surface area CD 4 Cells Cluster of differentiation cells Ct Cycle threshold value CYP gene Cytochrome P450 DHT Dihydrotestosterone DNA Deoxyribonucleic acid ECR Ethical review committee EIA Enzyme immune