EFFECTS of RECOMBINANT HUMAN INTERFERON-Α-2B on TESTICULAR MORPHOLOGY, TESTOSTERONE PRODUCTION and AROMATASE GENE in ALBINO RAT MODEL

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

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

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

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.

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

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 assay EO External oblique ESF External spermatic fascia FCV Feline calci virus FHV -1 Feline herpes virus FIP Feline infectious peritonitis FSH Follicle stimulating hormone GSI Gonadosomatic index HPT Hypothalamo pituitary testicular axis HSDH Hydroxysteroid dehydrogenase IFN Interferon IFNGR Interferon gamma receptor IO Internal oblique JAK Janus kinase LH Luteinizing hormone LSD Least significant difference MHC Major histocompatibility complex MIU Million international units NADPH Nicotine amide adenine dinucleotide phosphate NIH National institute of health NK cells Natural killer cells NSB Non specific binding OD Optical density PBS Phosphate buffer saline x

PCR Polymerize chain reaction PG Prostaglandin PS Primary spermatids PV Process vaginalis RNA Ribonucleic acid RS Round spermatids SCF Steel cell factor SD Standard deviation SHBG Sex hormone binding globulin SPSS Statistical package for social sciences STAT Signal transdusor and activator of transcription TMB Trimethyl benzedene TV Tunica vaginalis UTR Untranslated region

TABLE OF CONTENTS

Page # ACKNOWLEDGEMENT------iv ABSTRACT------v LIST OF ABBREVIATION------ix TABLE OF CONTENTS------xi LIST OF TABLES------Xiv LIST OF FIGURES------xv LIST OF GRAPHS------xvii

LIST OF PHOTOMICROGRAPHS------xviii

CHAPTER – I ------01

1. INTRODUCTION------01 2. OBJECTIVES ------04 3. RATIONALE OF STUDY------05 4. HYPOTHESIS------06

CHAPTER – II ------07

LITERATURE REVIEW ------07 1. Male reproductive system ------07 1.1. Parts of male reproductive system ------07 2. External genitalia ------08 2.1. Penis ------08 2.2. Scrotum ------08 2.3. Skin ------08 2.4. Superficial fascia ------10 2.5. Spermatic fascia ------10 2.6. Tunica vaginalis ------11 2.7. Lymph drainage ------11 3. Internal genitalia ------11 3.1. Epididymis ------11 3.2. Vas deferens ------12 3.3. Accessory glands ------12 4. Testis ------13 4.1. Structure ------13 4.2. Blood supply ------15 4.3. Lymph drainage ------15 5. Spermatic cord ------19 6. Testis development ------21 6.1. Descent of testis ------23 6.2. Congenital anomalies of testis ------25 7. Clinical conditions ------27 8. Histological structure of testis ------29 xii

8.1. Blood testis barrier ------29 8.2. Sertoli cells ------29 8.3. Leydig cells ------33 8.4. Spermatogenesis ------33 8.5. Mature spermatozoa ------37 8.6. Hypothalamo - pituitary – gonadal axis and hormonal control ------39 9. Steroid hormone ------43 10. Aromatase gene and aromatase ------43 11. Interferons ------49 11.1. Historical background ------49 11.2. Interferons ------50 11.3. Types of interferons ------51 11.4. Interferons producing cells ------52 11.5. Interferon receptors and signaling pathways ------53 11.6. Physiological effects of interferons ------54 11.6.1. Interferons as immune modulators ------55 11.6.2. Interferons as anti proliferative agents ------55 11.6.3. Interferons as inflammatory modulators ------56 11.6.4. Side effects of interferon therapy ------56 11.6.5. Recombinant Human IFN – ᾳ2a------57

CHAPTER – III ------58 MATERIALS AND METHODS ------58 1. Study Design ------58 2. Study Setting ------58 3. Duration of study ------58 4. Sample size ------58 5. Animal groups ------58 6. Sampling technique ------58 7. Sample selection ------59 7.1 Inclusion criteria ------59 7.2 Exclusion criteria ------59 8. Ethical committee approval ------59 9. Animal protocol and housing ------60 10. Experimental details ------60 11. Histology and photography ------60 12. Sperm morphology ------62 13. Testosterone assays ------62 14. Serum FSH assays ------67 15. Serum LH assays ------68 16. Aromatase gene detection ------70 17. Preparation of histological slides ------77

CHAPTER – IV ------80 RESULTS ------80

xiii

CHAPTER – V ------153 DISCUSSION ------153

CHAPTER – VI ------164 CONCLUSION ------164

CHAPTER – VII ------165 RECOMMENDATIONS ------165

REFRENCES ------166 APPENDIX------I APPENDIX------II

xiv

LIST OF TABLES No. Description Page

IV –1 Analysis of variance showing F- ratio and P value of various 84 study parameters in 80 rats models distributed into four equal groups ------IV – 2 Serum testosterone ------85

IV – 3 Serum FSH ------86

IV – 4 Serum LH ------87

IV – 5 Thickness of seminiferous tubule ------88

IV – 6 Diameter of seminiferous tubule ------89

IV – 7 Seminiferous tubule length ------90

IV – 8 Germ cell count in seminiferous tubules ------91

IV – 9 Germ cell maturation ------92

IV – 10 Seminiferous tubule desquamation ------93

IV – 11 Seminiferous tubule basement membrane ------94

IV – 12 Interstitial tissue ------95

IV – 13 Sertoli cell counts ------96

IV – 14 Leydig cell counts ------97

IV – 15 CT (cycle threshold) value in controls and experimental rats ---- 115

LIST OF FIGURES

No. Description Page

II – 1 Anatomy of spermatic cord , testis , Epididymis, vas deferens, 9 pampiniform plexus ------II – 2 Gross anatomy of testis and associated duct system------16

II – 3 Gross anatomy of testis and associated duct system ------17

II – 4 Lymphatic drainage of scrotum and testis------18

II – 5 Embryological development of testis and duct system ------22

II – 6 Processus vaginalis, its origin and development and descent of 23

ovary and testis ------

II – 7 Degrees of incomplete descent of testis ------24

II – 8 Few common congenital malformations of processus vaginalis 26

II – 9 (A) Hydrocele (B) Layers of scrotum traversed by needle for 26

tapping of Hydrocele ------

II –10 Transverse section of testis showing SC – sertoli cells, IC – 31

Interstitial cells of Leydig, GC- gametocytes, SP- spermatids,

SPC – spermatocytes ------

II – 11 Microscopic features of seminiferous tubule showing 32

spermatogenesis ------

II – 12 Steps of spermatogenesis within sertoli cells showing meiotic 35

and mitotic divisions in different stages of sperm production --

II – 13 The chromosomal number shown in different stages of sperm 36

production ------xvi

II – 14 Morphology of mature spermatozoa ------38

II – 15 Hypothalamo – pituitary – gonadal axis showing hormonal

control of testis and spermatogenesis ------41

II – 16 Functions of testosterone ------42

II – 17 Steroidogenesis pathway of testosterone synthesis ------44

II – 18 Steroidogenesis pathway of testosterone synthesis ------46

II – 19 Rate of aromatase enzyme in the testosterone conversion to 47

the estrogen ------

II – 20 Rate of aromatase enzyme in the testosterone conversion to 48

the estrogen ------

xvii

LIST OF GRAPHS

No. Description Page

IV – 1 Serum testosterone in different rat groups 98

IV – 2 Serum FSH in different rat groups 99

IV – 3 Serum LH in different rat groups 100

IV – 4 Thickness of seminiferous tubule in different rat groups 101

IV – 5 Diameter of seminiferous tubule in different rat groups 102

IV – 6 Total seminiferous tubular length in different rat groups 103

IV – 7 Germ cell counts in different rat groups 104

IV – 8 Germ cell maturation in different rat groups 105

IV – 9 Seminiferous tubule desquamation 106

IV – 10 Basement membrane thickness in different rat groups 107

IV – 11 Interstitial edema in different rat groups 108

IV – 12 Sertoli cell counts in different rat groups 109

IV – 13 Leydig cell counts in different rat groups 110

IV – 14 Bar graphs showing Ct value of gene expression 116

xviii

LIST OF PHOTOMICROGRAPHS

No. Description Page

IV – 1 Group I. Control – testicular tissue sections showing intact histological architecture, normal structure and normal 118 spermatogenesis. IV – 2 Group I. Controls – testicular tissue sections showing normal seminiferous tubules showing with intact histological 119 architecture. IV – 3 Group I. Controls – testicular tissue sections showing normal seminiferous tubules showing with intact histological 120 architecture. IV – 4 Decrease germ cell, maturation arrest, increased numbers of sertoli cells. 121 IV – 5 Thickened basement, increased Leydig cell, spermatogenesis arrest. 122 IV – 6 Thickened basement, increased Leydig cell, spermatogenesis arrest 123 IV – 7 Decreased germ cells, maturation arrest, thickened basement membrane, prominent clumping of epithelial cells with relatively 124 increased vascularity. IV – 8 Decrease germ cells, increased sertoli cells maturation, clumping of epithelial cells, thickened basement and arrest of 125 maturation. IV – 9 Decrease germ cells, increased sertoli cells maturation, clumping of epithelial cells, thickened basement and arrest of 126 maturation, relatively increased length of tubules and vascularity. IV – 10 Thickening of basement membrane relatively increased in vascularity, spermatogenesis is arrested, decreased germ cells, 127 increased Sertoli and Leydig cells, clumping of epithelial cells xix

and increased in the diameter of the tubules. IV – 11 Increased length, diameter, width of tubules, spermatogenesis is arrest, increased number of sertoli cells, increased number of 128 Leydig cells, increased vascularity and thickened basement membrane. IV – 12 Spermatogenesis is arrest, increased number of sertoli cells, increased number of Leydig cells, increased vascularity 129 increased thickened basement membrane with increased diameter of the tubules. IV – 13 Spermatogenesis is arrest, increased number of sertoli cells, increased number of Leydig cells, increased vascularity 130 increased thickened basement membrane with increased diameter of the tubules IV – 14 Spermatogenesis is arrest, increased number of sertoli cells, increased number of Leydig cells, and increased vascularity in 131 creased thickened basement membrane. IV – 15 Normal sperm count and morphology 132 IV – 16 Normal sperm count and maturation of sperms IV – 17 Normal sperm count and maturation of sperms 133 IV – 18 Normal sperm count and maturation of sperms 134 IV – 19 Normal sperm count, mixed premature and mature form of 135 sperm IV – 20 Normal sperm count with short tail with presence of primitive germ cell germinal epithelial cells 136 IV – 21 Normal forms of sperms with abnormal head and tail. 137 IV – 22 Normal sperms with premature form, plasma cells 138 IV – 23 Low sperms with premature form, plasma cells 139 IV – 24 Low sperm with predominantly premature forms of sperms 140 IV – 25 Low sperm with Premature and mature forms of sperms 141 IV – 26 Low sperm count with short tail with primitive germ cells 142 IV – 27 Premature forms of sperms 143 IV – 28 Abnormal forms of sperms with head and tail 144 xx

IV – 29 Abnormal sperms with premature forms and plasma cells 145 IV – 30 Abnormal and premature forms of sperms with plasma cells 146 IV – 31 Few mature sperms with predominantly immature forms of 147 sperm IV – 32 Primitive forms of sperms 148 IV – 33 Premature forms of sperms 149 IV – 34 Sperm with short tail 150 IV – 35 Premature forms of sperms and plasma cells 151

CHAPTER I

INTRODUCTION

Interferons (IFNs) are a family of biologically active proteins. They are secreted by the immune system. IFNs are produced by immune cells in response to viral and bacterial infections and/ or cancers. IFNs are divided into multiple types. However, the most common are IFN-α, IFN-β and IFN-γ (1).

Interferon’s (IFNs) are most potent biological cytokines of immune system, called immunomodulators. The IFNs exhibit biological effects like regulation of messenger RNA (mRNA) synthesis, cell division and cell growth, and exert antiviral activity, antibacterial activity and mediate immune regulation (2).The leukocyte IFNs are designated as IFN-α and IFN-γ, while fibroblast IFN are termed as the IFN-ß(3). It is reported that injecting IFN-γ in mice affects spermatogenesis and alters germinal epithelium (4).Transgenic male mice exposed to IFNs showed bizarre changes in spermatogenesis and eventually became sterile. Gonadal steroidogenesis is inhibited by IFN-γ in both in-vitro and in-vivo experiments, however the underlying mechanisms are not clearly elucidated (5-7).

In cultured cells,IFN-α is produced by peritubular myoid cells and Sertoli cells, as well as by germ cells. In contrast, it is evident that the IFN-γ is produced by early spermatids (8, 9).The IFN-α and -γ receptors are expressed on the membrane of sperm cells of mammals during sperm formation in the seminiferous tubules. The expression of IFN-α and IFN-γ receptors show that the IFNs may have implications in anti-sperm vaccine contraception and male

2 infertility. Gene mutations in controlled targeted studies have shown IFN inhibit development of germ cells in testes (3, 10).

It is observed that the over expression of IFN α and/or IFN-ß gene disrupts the spermatogenesis and eventual destruction of spermatogonia in the transgenic mice testes mice, as reported by previous studies (5, 6). Serum testosterone and free androgens are reduced when IFN-α was administered in healthy male (11, 12). Previous studies have reported that IFN–γ induces deleterious effects on testes histology with desquamation of germinal epithelium, and height of germinal cells, seminiferous tubule diameter and

Sertoli cells are all reduced (3, 10).

The effects of IFN-α on testicular morphology and functions have never been clearly elucidated. Animal studies have shown conflicting results. In one study, the IFN-α expression in transgenic mice showed degeneration of spermatogonia and atrophy of seminiferous tubules (11).Other studies reported that the IFN-α when administered subcutaneous tissue, it expanded the spermatogenesis and serum testosterone levels were elevated. (13,

14).The testosterone plays an important role in the spermatogenesis directly and indirectly through conversion into estrogen. The estrogen is essentially involved in sperm maturation in their terminal stages. The testosterone is converted into estrogens by the aromatization, the reaction being catalyzed by the action of p450-aromatase enzyme. The aromatase enzyme (AE) is expressed in the testis, liver and brain tissues. The AE expression has been affected by different stimuli such as the morphine, and the recombinant human interferon-α-2b (rh-INF-α-2b) which needs to be investigated also (15).

IFNs administration and effects on several body systems have been extensively investigated. However, only a few studies on the controlled use of

IFNs are available on reproductive system. Moreover, most of the work in literature, concerning effects of Interferons addresses the hormonal, and more generally the homeostatic effects of these drugs. The effects of these drugs on the phenotype of testicular histology have never been researched before. As currently Pakistan has much burden of viral hepatitis for which ideal curable drug being in use is the recombinant human interferon-α-2b (rh-INF-α-2b), but its effects on testicular morphology, hormone production and aromatase gene are not evaluated in any study. Hence, the present study was therefore proposed to determine the phenotypic and genotypic effects of rh-INF-α-2b on testicular morphology, hormone production and aromatase gene in adult rat model.

AIMS/OBJECTIVE OF STUDY

The objectives of present research study are:

1. To investigate the phenotype 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 androgenic hormone

production.

4. To examine the effects of rh-INF-α-2b on hypothalamo-pituitary-gonadal

(testicular) axis

5. To study the effect on rh-INF-α-2b on the aromatase gene.

RATIONALE OF STUDY

Currently, recombinant human interferon-α-2b is widely used as therapy in Pakistan particularly for the treatment of viral hepatitis beside of its known side effects. The side effects are reported to occur on each tissue, organ and systems of body including effect on testicular structure and functions. Present study will help to provide basic concepts on sexual dysfunction caused by recombinant human interferon-α-2b therapy.

HYPOTHESIS

The effects of recombinant human interferon-α-2b will be evaluated on testicular morphology and hormone production.

Null Hypothesis [Ho]: will be accepted true if no change in testicular morphology, hormone production and aromatase gene is observed in rats after injections of recombinant human interferon-α-2b.

Alternative Hypothesis [H1]: will be accepted true if Null hypothesis is rejected i.e.; a change in testicular morphology, hormone production and aromatase gene is observed in rats after injections of recombinant human interferon-α-2b.

CHAPTER II

LITERATURE REVIEW

1. MALE REPRODUCTIVE SYSTEM

The male reproductive system comprises of gonads, its duct system

and male sex glands. The male gonads are a pair of testes enclosed within

scrotum which is an out pouching of anterior abdominal wall structures.

The scrotum lodges testes hanging out of pelvic cavity to maintain

optimal scrotal temperature necessary for spermatogenesis. The male organs

are necessary for sexual intercourse, sperm ejaculation, necessary to fertilize

an ovum into zygote which eventually develops as an embryo and fetus.

1.1. Parts of Male reproductive system

 External genitalia o Penis o Scrotum

 Internal genital organs o Testes o Epi-didymis o Vas-deferens o Ejaculatory-duct o Accessory glands . Prostate-gland . Seminal-vesicles . Bulbo-urethral glands (16 - 19)

2. EXTERNAL GENITALIA

2.1. Penis

The male copulatory organ is penis necessary for sexual intercourse.

The penis has a long shaft with a flat bulb like tip called the glans penis. The penis contains erectile spongy tissue to hold blood during erection. The venous sinuses with erectile spongy tissue become filled with blood to make penis erectile. The arteries are dilated during erection and veins are compressed.

2.2. Scrotum

Anterior abdominal wall has an out pouching called scrotum which holds and protects testes. In adult life, scrotum is connected to pelvic cavity through inguinal canal. The scrotum contains two muscles; the cremaster and dartos muscles. The cremaster pulls scrotum close to abdominal wall and dartos muscles makes scrotum wrinkled. The cremaster and dartos relax during hot season and contract during cold season.

Scrotum contains Epididymis, testes and terminal ends of spermatic cords. Anatomically, scrotal wall shows following layers;

2.2.1. Skin Scrotal skin is naturally wrinkled like a thin paper. It is pigmented skin which hangs out as a pouch. Scrotal skin shows a midline ridge which is a remnant of embryonic fusion of labio-scrotal swellings.(16 - 19)

Figure II-1: Anatomy of spermatic cord, testis, Epididymis, vas deferens, Pampiniform plexus. Adapted from; SNELL (17). Page; 129.

2.2.2. Superficial fascia

The superficial fascia is a membranous but lightly fatty layer of anterior wall of the abdomen. Within the scrotum, the fatty layer is replaced by

“involuntary smooth muscle” layer which is anatomically known as the “dartos muscle”. Dartos muscle gets sympathetic nerves which are involuntary. The wrinkling of skin is under control of sympathetic autonomic nerves. Colle`s fascia (membranous layer) is a continuity of the Scarpa’s fascia of anterior wall of the abdomen. This fascia is attached to the perineal body on its posterior side and to the edge of the membrane of the perineum too. Along the edges, it is connected to the “ischio-pubic rami”. Two layers of superficial fascia divide it into 2 partitions which cross through the scrotum and isolate the testes.(Figure

II-1)(16 - 19)

2.2.3. Spermatic fasciae

This fascia has three layers. They lie under the superficial fascia. They are in continuity with the 3 layers of the anterior wall of the abdomen. External oblique (EO) muscle aponeurosis continues as external spermatic fascia

(ESF), internal oblique (IO) as cremasteric fascia and transversalis muscle fasciacontinues as fascia-transversalis.

Genital branch of genitor-femoral nerve innervates the cremaster muscle. If one strokes medial aspect of thigh with sharp edged instrument, the cremaster muscle contracts and this forms basis for checking the cremasteric reflex.

Sensory nerve fibers of cremaster reflex arc run through the L1 and L2 which are the fibers of the femoral branch of genitor-femoral nerve. While the efferent motor fibers are running through the genital branch of the “genitor- femoral nerve”. Cremaster muscle contraction raises the testis and the scrotum upward for making scrotal temperature regulated and protects against injury(Figure II-1) (16 - 19).

2.2.4. Tunica vaginalis (TV)

TV is contained within the “spermatic fasciae”. TV encroaches onto the anterior, lateral and medial surfaces of respective testes. Processus vaginalis is lower expanded end of TV. Soon before birth, the communication between

Processus vaginalis and TV is closed. TV is a closed sac which is invaginated from inside by testes (Figure II-1)(16 - 19).

2.2.5. Lymph Drainage

The skin, tunica vaginalis and spermatic fascia drain lymphatics into the superficial inguinal lymph nodes (Figure II-1)(16 - 19).

3. INTERNAL GENITALIA

3.1. Epididymis

The Epididymis is about 6 metres in length in human beings, 1 meter in mouse and 3 meters in rats. The Epididymis is connected to testes by a fold of serous membrane. In adult life, the Epididymis is divisible into 3 regions:

 Globus major (Head) upper elongated part. Efferent ductules connect it

with upper end of testis.

 Body is the central part

 Globus minor (tail) lower pointed part. The tail of Epididymis is connected

with the ductus deferens (vas deferens).

3.2. Vas deferens

The vas deferens is approximately 25 centimeter long thin tube extending from Epididymis to pelvic cavity.

3.3. Accessory glands

The accessory glands are three in number;

 Seminal vesicles

 Prostate gland,

 Cowper glands (Bulbourethral glands)

The accessory glands secrete fluid rich in nutritional substances and provide lubrication(16 - 19).

i. Seminal vesicles

The seminal vesicles are corrugated sac like structures. They lie on back surface of urinary bladder. It is attached to vas deferens at one end. They add to semen a sticky, yellow secretion rich in fructose. About 70% of semen is added by seminal vesicles. The secretion of seminal vesicles is rich in

13 fructose, prostaglandins, fibrinogen and citric acid. The fructose provides energy to spermatozoa and aids in spermatozoa motility while prostaglandins help in increasing female tract motility and aids in fertilization of ovum.

ii. Prostate gland

The prostate gland surrounds the ejaculatory duct and male urethra at the base of urinary bladder. The secretion of prostate gland makes semen milky in color. The prostate glands add to semen a fluid rich in calcium, phosphate, citrate and profibrinolysins. The prostate fluid is alkaline in nature, which is important for fertilization of ovum. The alkaline fluid increases sperm motility and fertility.

iii. Bulbourethral glands

The Bulbourethral glands (Cowper’s glands) are small pea-sized structures located just below prostate gland on the sides of urethra. The glands secrete a clear slippery fluid directly into urethra. The secretions serve to lubricate urethra and neutralize acidity of urine in the urethra(16 - 19).

4. TESTIS

4.1. Structure:

The testes are ovoid structures about 5 centimeters long which lie suspended by spermatic cord within the scrotum. They contain about 200-300 lobules which are separated by connective tissue septa. Each lobule contains

2-3 seminiferous tubules which are highly coiled and are about 1 meter in

14 length. The seminiferous tubules discharge contents into rete testis. The testes are covered by tunica albuginea and tunica vaginalis. The tunica vaginalis is distinguishable into visceral and parietal layers similar to peritoneum of abdominal cavity. The testes receive arterial blood through testicular artery and venous drainage goes through pampiniform plexus. (Figure II-2, II-3) (16 -

19). The testes are mobile lying within the scrotal sac. They are firm to touch.

Right testis lies slightly above the left testis. A tough fibrous capsule surrounds each of the testes called “tunica albuginea”. Fibrous tissue septa separate testes into small lobules. Each lobule lodges 1-3 coiled structures called

“seminiferous tubules”. Rete testis is the final opening place where tubules open into a network of channels. Rete testis is connected to the upper end of the “epididymis” through small ductules known as the “efferent ductules’.

Spermatogenesis occurs typically at temperature slightly lower than that within the peritoneal cavity. A testis lying in scrotum is typically having a temperature of 3°C less of body temperature which is optimal for spermatogenesis (16 - 19). The temperature sensing and its control in testes is not fully understood, but the reflex actions in scrotal skin under the pressure caused by dartos and cremaster muscles might be some explanations. One logical proposed mechanism is that there is working of a counter current exchange system between veins and arteries within spermatic cord. As the veins are lying close to arteries, the heat dissipates from arteries to veins and this helps balancing the scrotal temperature. By this infers, the hot blood connecting in the course from the mid loses warmth to the blood ascending to inside the veins.

4.2. Blood Supply

Arising from abdominal aorta is the branch called testicular artery which supplies arterial blood to testes. Pampiniform plexus is a venous network which originates in epididymis and testes and continues upward as testicular veins. The vein of right testis is tributary of inferior vena cava. While the left testicular vein drains into left renal vein.

4.3. Lymph Drainage

Lymphatics travel through spermatic cord to drain into lumbar and para

– aortic lymph nodes. The lumbar and para – arotic lymph nodes lie at the level of transpyloric plane (Figure II-4)(16 - 19).

F Figure II. 2. Gross anatomy of testis and associated duct system. Adaptedfrom: Tank, 2009. (18)

Figure II.3. Gross anatomy of testis and duct system. Adapted from: Tank, 2009. (18)

Figure II-4: shows lymphatic drainage of scrotum and testes. Adapted from; Snell, 2012. Page; 130. (17)

5. SPERMATIC CORD

The spermatic cord is a thick cord like structure for passage of structures from and to the testes through the inguinal canal. It starts at the

“deepinguinalring” of inguinal canallying laterally to inferior epigastric artery and finishes at the testis. (Figure II-1, II-2)

Structures of the Spermatic Cord

The structures are as follows:

 Remnants of the “Processus vaginalis”

 Genital branch (L1, L2) of genitor-femoral nerve fibers

 Autonomic nerve fibers

 Testicular artery

 Testicular veins are running in form of a plexus known as the

Pampiniform plexus

 Vas-deferens (Ductus deferens)

Vas Deferens (Ductus Deferens)

Vas deferens is a thick cordlike. It is thick and muscular duct. It is palpable in between thumb and middle finger if felt within the scrotum. It main function is transportation of sperm from epididymis to urethra.

Testicular Artery

It is a branch of the “abdominal aorta”. Bony mark of origin lies at the

2nd lumbar vertebra. It is courses on the posterior abdominal wall. It is thin and long artery. It reaches to testis by passing through the inguinal canal. It supplies blood to the testes and epididymis (16 - 19).

Testicular Veins

Pampiniform plexus an extensive interwoven network of veins which leaves the posterior border of the testis. It forms a single testicular vein at the deep inguinal canal ring as it ascends up. Pampiniform plexus forms testicular vein eventually, which drains into the left renal vein (Figure II-6).

Lymph Vessels

Testicular lymphatic ducts pass through the inguinal canal. Passing through the posterior abdominal wall and reach the lumbar (para-aortic) lymph nodes on the side of the aorta at 1st lumbar vertebra.

Autonomic Nerves

The sympathetic nerves accompany testicular artery after originating from nerve plexuses. Sensory nerve fibers also travel through sympathetic nerve fibers.

Processus Vaginalis

Processus vaginalis remnants are present within the “spermatic cord”.

Genital Branch- of Genito-femoral Nerve

Cremaster muscle is supplied by L1 through genital branch.

(16 - 19)

6. TESTIS DEVELOPMENT

There are two phases in the developing testes; first during embryonic life and later on in pre-pubertal period.

During embryonic period, the primitive gonads are capable of becoming either testes or ovaries and are called bi-potential gonads. In human beings, beginning at 4th week, the bi-potential gonads are present within intermediate mesoderm close to developing meso-nephros. At 6th week, sex cords develop within the bi-potential gonads forming testes. At this stage, gonads show primitive Sertoli cells which surround germ cells that have migrated into gonads soon before sex differentiation. Male gonad development is dependent on SRY gene of Y-chromosome. The SRY gene produces TDF (testes determining factor) which differentiate primitive gonads into male testis. The

SRY gene stimulates other sex determining factors such as GATA4, SOX9 and AMH. These sex determining genes and factor cause male phenotype of including directing development of the early bi-potential gonad down the male path of development. Testes follow the "path of descent" from high in the posterior part of fetal abdomen to the inguinal ring and beyond to the inguinal canal and then descending into the scrotum. Complete descent of testes normally occurs in most cases (97% full-term, 70% preterm) by birth.

During pubertal period, the testes begin growing at the onset of spermatogenesis. Size of testes at this point depends on sperm production, interstitial fluid, and fluid production by Sertoli cell (Figure II-5)(16 - 19).

Figure II-5: Embryological development of testis and its duct systems. Adapted from; Snell, 2012. Page; 134 (17).

6.1. Descent of the Testis

The testis grows high up on the posterior abdominal wall. It descends to peritoneum posteriorly in late fetal life. During its descent, it retains its blood supply, nerve supply, and lymphatic’sof its origin.Processus vaginalis pulls it down though a particular anatomical path as shown in FigureII-6. (17)

Figure II-6: Processus variganlis- its origin, and development and Descent of Ovary and testis. Adapted from; Snell, 2012. Page; 134 (17)

Figure II-7: Degrees of incomplete descent of testis as indicated by numbers1-4. Adapted from; Snell, 2012. Page; 134 (17)

6.2. Congenital Anomalies of the Testis

Following congenital anomalies may occur in developing testes.

 Anterior inversion- Testes and its layers lie posteriorly while epididymis is

seen lying anteriorly.

 Polar inversion, both the epididymis and testes are inverted completely.

 Imperfect descent (cryptorchidism): is defined as incomplete descent. A

testis may lie in its path of normal descent but never reaches to scrotum. A

testis may be impacted in inguinal canal, within superficial ring, or upper

part of scrotum. Testis may lie within abdominal cavity.

 Maldescent is characterized by an aberrant descent pathway. Testis never

reaches to proper destination. Testis may be found in

o above the inguinal ligament,

o in the pubis anteriorly,

o in the perineum,

o Or may bewithin the thigh.

Many authorities had reported of an increased of incidence of neoplastic changes in the undescended testes.

The “appendix” of the testis and of the Epididymis is embryologic leftovers located at the upper anatomical pole and may become cystic. The

“appendix” of the testis originated from the “para-mesonephric duct“. While the

“appendix”of epididymis represents remnant of the “mesonephric tubules”(Figure II-7) (16 - 19).

Figure II-8:Shows few common congenital malformations of Processus vaginalis. Adapted from; Snell, 2012. Page; 132 (17)

A B

Figure II-9:A.Fluid tunica vaginalis (Hydrocele). B. Layers of scrotum traversed by needle for tapping of Hydrocele. Adapted from; Snell, 2012. Page; 133 (17)

7. CLINICAL CONDITIONS

Varicocele

When the “pampiniform venous plexus” becomes elongated, tortuous and engorged of blood is called “varicocele”. Most commonly occurs on left side of body because left testicular vein enters at an obtuse angle into renal vein. Also the left renal vein is high pressure vessel compared to inferior vena cava. Young adults and adolescent age is commonly affected. An acute varicocele on left side may occur because of invasion of renal vein by renal cell carcinoma (17).

Testicular tumor

Malignant testicular tumor is a cancer of testicular tissue. It may originate from germ cells or non germ cells. Metastasis through lymphatics reaches at the lumbar / paraortic lymph nodes located at L1level.The tumor spreads and invades the scrotla skin involving superficial inguinal lymph nodes.

Testicular torsion

Rotation of testis around spermatic cord within scrotum results in torsion of testis. A large lax tunica vaginalis makes testis prone to torsions.

Young male and children are often affected with sudden onset of excruciating pain. If the condition is not managed properly and prompt, it may result in complete cessation of blood supply and complete testicular necrosis(17).

Processus Vaginalis (PV)

Typically, the upper segment is wrecked just before birth while the lower portion stays as the tunica vaginalis. The PV is liable to the accompanying inherent abnormalities:

1. It may participate partially or entirely as a “preformed hernia sac”making an

“indirect” inguinal hernia.

2. It is narrowed down but communicates well with peritoneal cavity. Taking the peritoneal fluidand resulting in acongenital hydrocele.

3. The upper and lower closures of the processus may get to be devastated, yet leave a little cystic region in the center which turns into an encysted hydrocele of the cord.

The tunica vaginalis is attached to the testicular sides. Thus, it is common to get fluid inside of this tunic. This is suggested pretty much as a hydroceles. Hydroceles are mostly of “idiopathic” nature.To expel overabundance liquid from the tunica vaginalis is surgically called tapping a hydrocele. It is generally performed with cannula and a fine trocar which are embedded across the scrotal skin. The structures crossed by cannulainclude;

o Skin,

o Dartos

o Colles’ fascia

o External spermatic fascia,

o Cremasteric fascia,

o Internal spermatic fascia, and

o Tunica vaginalis- parietal layer. (Figure II-8 and II-9) (16 - 19)

8. HISTOLOGICAL STRUCTURE OF TESTES

The testis internally, comprises wavy long tubules called seminiferous tubules where spermatozoa are formed from mitosis of primordial germ cells.

Their epithelium is lined by Sertoli cells. They also support the developing sperms.Spermatids are released into the tubular lumen at the end of spermatogenic cycle, and are then transported to the head region of epididymis for maturation.

8.1. The Blood-Testis Barrier

Spermatogenesis is a vulnerable process and protection of germ cells against noxious influences. Part of this protection is mediated by the BTB that physically separates the meiotic germ cells from the blood circulation. The

BTB is created by the Sertoli cell process. These processes divide the seminiferous epithelium into the basal and the ad-luminal parts respectively.

However, the peri-tubular and endothelial cells also contribute to the function of the barrier (Figure II-10) (16 - 19).

8.2. The Sertoli Cell

The Sertoli cells line epithelium of seminiferous tubules and are critical for spermatogenesis. Sertoli cells nurse, nurture and protect developing spermatids. The Sertoli cells provide nutrients like sugars, amino acids, vitamins, minerals and lipids to developing spermatids. The Sertoli cells form blood testis barrier which is essential for developing spermatids, thus two compartments are formed; viz an ad luminal compartment and a basal compartment. The later carries spermatogonia and early spermatocytes, while

30 the other one carries spermatocytes and spermatids. Cytoskeleton maintains the cell shape. The primary components of cytoskeleton, the microtubules are affected by certain toxicants, telling their role in sperm production.

Microtubules lye along the length of Sertoli cells, and allow anchorage of germ cells, and translocation of developing stages of germ cells from basal to ad luminal regions(Figure II-10, II-11)(16 - 19).

Sertoli cell functions

 Spermatogenesis regulation- through release of FSH and testosterone

 Sertoli cells sequester the meiotic and post-meiotic germ cells

 Produce advertisement luminal parts which are confined from both

serum and lymph

 Attachment of germ cells through one of a “fine intermediate” fiber

(desmosome-like intersections) and microfilament (actin-ectoplasmic

specializations, ESs) intersections;

o Sertoli cells anticipate to slough the immature “germ cell” from the

germinal epithelium of the seminiferous tubules.

o Presence of desmosome-like junctions from the very beginning

o Ectoplasmic specializations then supplant this intersection

(20, 21)

Figure II.10: Transverse section of testis showing; SC-sertoli cells, IC-Interstitial cells Leydig, GC-gametocytes, SP-spermatid, SPC-spermatocyte. Adapted from: (16)

Figure II.11.Microscopic features of seminiferous tubule showing Spermatogenesis. Adapted from: GUYTON, et al. (21)

8.3. Leydig Cells

Leydig cells are so called in the remembrance of its inventor; a German

Zoologist Franz von Leydig (1821 - 1908). The Leydig cells are lying in the interstitial space between seminiferous tubules. The Leydig cells produce male androgen hormone.

 Fetal Leydig Cells

During fetal period, they produce male androgenic hormones, which are critical for differentiation of male genitalia. The fetal Leydig cells are dependent on pituitary luteinizing hormone (LH) and adrenocortical adrenocorticotrophic hormone (ACTH) for androgen synthesis.

 Adult Leydig Cells

Adult Leydig cells show lipid droplets in cytoplasm. Leydig cells have well developed agranular endoplasmic reticulum. Mitochondria show tubular cristae. They are actively engaged in the steroid biosynthesis. The cells also show Lipofuscin granules, lysosomes and Reinke’s crystals.(16 - 21) 8.4. Spermatogenesis

Amid embryonic period, the primordial germ cells move to primitive gonad (testes) and get to be juvenile germ cells called as spermatogonia. The spermatogonia lie in the layers of inside surface of seminiferous tubules.

Mitotic division begins in spermatogonia onset of puberty and mature spermatozoa are produced through continual proliferation and meiotic division.

Formation of spermatozoa is known as Spermatogenesis. The process of spermatogenesis begins at the beginning of the puberty of a male. It occurs within the seminiferous tubules. Hypothalamus is the seat of onset of puberty.

It controls the gonadotrophins of anterior pituitary gland which have influence on the induction spermatogenesis and spermatogenesis. The average age of puberty onset is about thirteen years of age. During the primary phase of

“spermatogenesis”, the spermatogonia move among Sertoli cells toward the focal lumen of the seminiferous tubule. The Sertoli cells are expansive; with flooding cytoplasmic envelopes that encompass the newly developing spermatogonia the distance to the focal lumen of the tubule. They undergo mitotic and meiotic divisions. The spermatogonia which penetrate into Sertoli cell layer become developed into large primary spermatocytes. Each of primary spermatocytes in turn undergoes meiotic division to produce secondary spermatocytes. Over next few days, the secondary spermatocytes divide to produce spermatids. The spermatids undergo meiotic division to form mature spermatozoa. During development from spermatocytes to spermatids stage, the 46 chromosome are halved so that 23 chromosomes go to each spermatid. The steps of spermatogenesis and chromosome distribution are shown in figure II-4 and 5. (16, 20, 21)

Figure II-12. Steps of spermatogenesis within Sertoli cells and meiotic and mitotic divisions are shown in different stage of sperm production. Adapted from www.wikipedia/malespermatogenesis/15thMarch2015.

Figure II-13. The chromosome number is shown in different stages of sperm production.Adapted from www.wikipedia/malespermatogenesis/15thMarch2015.

8.5. Mature Spermatozoa

The mature spermatozoa are composed of a head, neck and tail. Head comprises of condensed genetic material, covered over by a thick membrane called acrosome. The acrosome contains enzymes necessary for penetrating the connective tissue surrounding the ovum to be fertilized. The enzymes of acrosome include hyaluronidase, acrosin and other proteolytic enzymes. The neck is loaded with mitochondria to supply energy for sperm flagella motility in form of ATP. The flagellum comprises of cytoskeleton. The flagellum contains central skeleton of 11 microtubules (axoneme). The cytoskeleton is critical for the sperm motility. The different parts of sperm cells are shown in figure II-

6.(20, 21)

Figure II.14. Morphology of mature spermatozoa. Adapted from www.wikipedia/malespermatogenesis/15thMarch2014.

8.6. Hypothalamo-pituitary-gonadal axis and hormonal control

The hormones of hypothalamus, anterior pituitary and Leydig cells of testes are all controlling the process of spermatogenesis. The hypothalamus and related organs controlling spermatogenesis and other testicular functions are collectively known as hypothalamo-pituitary-gonadal axis. (Shown in

Figure II.7) These hormones provide hormonal support necessary for growth, differentiation and maturation of sperm cells and also provide nutrition indirectly by stimulation accessory glands of male reproductive system:

1.Testosteroneis male androgen hormone secreted by the Leydig cells. The testosterone is essential for immature spermatocytes proliferation and growth, which is the first stage in forming sperm.

2.Luteinizing hormone (LH), is secreted by APG under the influence of hypothalamus. LH induces testosterone secretion by Leydig cells.

3.Follicle-stimulating hormone (FSH), also secreted by the anterior pituitary gland under stimulation from hypothalamus, stimulates the Sertoli cells. The

Sertoli cellsconvert spermatids to grow into sperm (the process of spermiogenesis). The spermatogenesis occurs solely under nursing of Sertoli cells.

4.Estrogens: Estrogens are produced from testosterone by the Sertoli cells by aromatization reaction catalyzed by aromatase enzyme. The reaction is controlled by FSH, and is essential for spermiogenesis.3

5.Growth hormone, thyroxine, insulin, cortisol and many others are hormones control the metabolic functions of testicular cells which are essential for the spermatogenesis (22, 23).

Figure II.15. Hypothalamo-pituitary-gonadal axis showing hormonal control of testes and spermatogenesis.Adapted from: WEINBAUER (23)

Figure II-16.Functions of testosterone. Adapted from:WEINBAUER, GF(23)

9. STEROID HORMONES

Testosterone is the main male androgen hormone along with minor fractions of 5α-dihydrotestosterone (DHT), androstenedione, androsterone, 17- hydroxyprogesterone, progesterone and pregnenolone. The testosterone plays a pivotal role in spermatogenesis. The lack of testosterone, Leydig cells and

Sertoli cells produces severe impairment of spermatogenesis. However, the role of progesterone, androsterone, and 17-hydroxyprogesterone in the testis is unknown (24, 25).Testosterone is metabolized to 5α-dihydrotestosterone

(DHT) by testicular 5α-reductase enzyme and converted to estradiol by aromatase enzyme. What is role of these later in spermatogenesis need to be elucidated? Because 5α-reductase enzyme inhibitors as such produce no defect in spermatogenesis. However, 5α-reductase enzyme inhibitors administration had produced defects in spermiogenesis in animal models. (20,

21, 23)

Figure II-17. Steroidogenesis pathway of testosterone synthesis. Aromatase enzyme is also shown.Adapted from: LU, W.(22)

10. AROMATASE GENE AND AROMATASE

The CYP19A1 gene is responsible for the coding of the aromatase enzyme. The location of this gene is detected at the 15q21 of the humans.

This gene spans 120 kb and it has a large and complex upstream regulatory unit comprising un-translated exon I and a number of promoters. It also carries a 30 kb coding region consisting of 9 translated exons II to X. (26)

Untranslated exon I is associated with ten different “tissue specific” promoters which is least quoted number. As the exon I is never translated hence the coded and translated protein is similar in most of the tissues. Each tissue utilizes its own promoters and associated enhancers and suppressors to express a unique untranslated first exon 5’-UTR (27 - 29). This differential splicing process leads to different amounts of mRNA transcripts, differences in mRNA stability and protein translation. As a result, this process defines the regulation of tissue specific aromatase activity for the estrogen biosynthesis

(27, 30, 31).

10.1. Cytochrome P450 system and Aromatase enzyme

The cytochrome P450 (CYPs) are a superfamily of enzymes. The CYP- enzymes contain a heme molecule non-covalently attached to a polypeptide chain. The CYPs use oxygen and hydrogen (H+) ion. The H+ is donated by reduced NADPH for oxidizing the endogenous and exogenous compounds like bile acids, steroids, drugs, chemicals and other xenobiotics. The aromatase is a key enzyme of sex steroidogenesis in the gonads. The CYP which catalyzes reaction of demethylation and aromatization of testosterone into estradiol and androstenedione to estrone is known as the aromatase enzyme.(22)

Figure II-18. Steroidogenesis pathway of testosterone synthesis. The Aromatase enzyme is also shown. Adapted from: LU, W. (22)

Figure II-19. Role of aromatase enzyme in the testosterone conversion to estrogens is shown. Adapted from:LU, W. (22)

Figure II-20. Role of aromatase enzyme in the testosterone conversion toestrogens are shown. Adapted from: VAZ, et al.(32)and LU, W. (22)

11. INTERFERONS

11.1. Historical Background

Isaacs and Lindenmann (1957) induced experimental infection into chorioallantoic membrane cultures of chicken embryos with influenza A-virus.

He reported a nonviral protein that induced resistance to host cells against influenza A-virus and other superinfecting viruses (33). Although, Nagano, et al(34) reported this protein before, but Isaacs and Lindenmannnamed it interferon (IFN) (33), because of its role in interfering the viral infections.

IFN discovery was a hope as a potent antiviral agent. However, this protein occurs in very meagre volumes and its purification was years when its purification and characterization was performed (35, 36). Molecular biotechnology, particularly the recombinant DNA technology, made it possible to be produced in enough amounts for research therapeutic reasons. Several human IFN-α (hIFN-α) subtypes were cloned in 1981 by WECK, et al(37), followed by the cloning of human IFN-β (hIFN-β) (38), and murine IFN-γ (m

IFN-γ) (39).

Human interferons exerted antiviral effects against herpes simplex keratitis when administered alone (40 - 42) or in combination with nucleoside analogs (43 - 45). In early HSV-1 infections, the topical application of

IFNreduced severity and duration of and shortened the period of virus shedding (46).

Natural and recombinant human IFNs were found effective antiviral agents in feline viruses in vitro. HuIFN and/or FeIFN inhibited the effects of feline leukemia virus (47), rHuIFN-α-2b co-administered with feline IFN inhibited FHV-1 and FCV (48, 49). Recombinant hFN-α and FeIFN-ß had similar antiviral activity against feline infectious peritonitis (FIP) (50). Previous

“in-vivo” and “in-vitro” research studies were conducted to determine the effects of rHuIFN-α combined with anti-viral drug chemotherapy (49, 51). For

FHV-1, Weiss (49) demonstrated strong synergism between ACV and rHuIFN-

αA/D hybrid in vitro. Similar reports were found with other clinical trials of HSV-

1-treatment in humans (43).

11.2. Interferons (IFNs)

Interferons (IFNs) are mediators and modulators of immune system.

IFNs are biologically active peptides known as cytokines (33). The cytokines, including IFNs, mediate physiological interactions among cells of immune system to function properly.

The cells of immune system are peripatetic in localization, not located at a single specific organ. In other words, inflammatory stimuli are detected first by a set of cells collectively known inflammatory cells. They help clear the stimuli, repair tissue damage and stimulate immune system. Specific inflammatory cells present antigens to lymphocytes of immune system, and are collectively known as antigen presenting cells (APC). Immune cells are highly dynamic cells which co-ordinate physically with each other. This co- ordination is made possible only through immune cytokines like IFNs. The

IFNs are of unique characteristics. IFNs communicate the immune cells and

they co-ordinate with other immune cells and cytokines, acting even at the

remote areas. Hence they are called specific messengers which may act

locally or generally. Unique characteristics of IFNs make them an ideal and

perfect mediators and modulators of the immune system. Some typical

characteristics are as follows;

i. Most cytokines are peptides of small size

ii. A singly cytokine is synthesized and secreted by different cells iii. A singly cytokine may act at different cells of immune system and

inflammation both. iv. Cytokines share similar physiological functions

v. Physiological functions are mediated as a cascade of reaction hence one

cytokine can stimulate many target cells. vi. The cytokines produce “synergistic” and/or “antagonistic” effects.

The Interferons are important mediators and modulators of immune

system, equally contributing in health and disease (33, 51, 52)

11.3. Types of Interferons (IFNs)

Interferons (IFNs) are broadly classified into three types, but this

classification is based on specificity of the “cell receptor binding”, structural-

homology and the biological activity. Types are as under;

 Type I IFN:

Type I IFNs have many family members. Of these, 2 are very important and used widely. These 2 include the; IFN-α and IFN-β (51, 52)

 Type II IFN:

Type II IFNs are designated as interferon-gamma (IFN-γ) or immune interferons. Type II IFNs use different types of receptors and physiological functions are also distinctively different.(53)

Type I IFNS bind to the similar type of receptors of cell. They elicit the physiological effects through similar signaling pathways and similarly regulate the expression of similar biological activities (54, 55). But other reports show distinct biological activities of IFN-α and IFN-β (55).

 Type III IFN: Type III include interferon lambda (IFN-λ) but it has not yet

been used clinically. (56 - 58).

11.4. Interferon producing cells

Virtually all of the human body cells are capable of producing and secreting the type I-IFNs. The body cells produce IFNs (IFN-α/β) in response to viral infections. The main source is however, the macrocyte-macrophage system. The antigen presenting cells of monocyte-macrophage system are highly equipped to release the type I Interferons. Gamma type IFNS (IFN-γ) are produced and secreted by the monocyte-macrophage system, the NK

(Natural killer) cells, and T- cell lymphocytes; mainly CD4+ type 1 T helper cells. As the IFN-α is a powerful activator of monocyte-macrophage system, the NK (Natural killer) cells, and T- cell lymphocytes; mainly CD4+ type 1 T

53 helper cells, hence indirectly accelerates the synthesis and secretion of IFN-γ.

The IFN-α increases the synthesis and secretion of IFN-β too. (59 - 61)

11.5. Interferon receptors and signaling pathways

Interferon receptors are trans-membrane proteins, which transverse the whole thickness. The receptors are located on the cell membrane surface and extend to other side reaching to cytoplasm, forming a cytoplasmic tail. (51)The

IFN receptors are classified into two classes;

- IFN receptors Class I – comprise of 2 subunits termed as the “α-” and

“β-” subunits which are called the “IFN-αR1” & “IFN-αR2” receptors.

- IFN receptors Class II – comprise of 2 peptide chains of IFNGR1 and

IFNGR 2.

The signaling pathway is the sequence of events "which produce biological effects of interferons. The signaling pathways include the Janus kinases and STATS.

Janus kinases: Janus kinases include the JAK 1, JAK 2 and TYS 2 members.

The 3 Janus Kinase acts as “enzyme”, which help in donating PO4-groups to the concerned substrates for their activation. As these are receptor bound, hence once stimulated by binding of IFNs, they produce the biological effects

(62, 63).

STATS: They are “signal transducer” which activates the process of the transcription. The STATS produce biological effects through gene transcription. Seven mammalian STAT proteins are known, however, only two

STAT 1 & STAT 2 are involved for the biological functioning of the IFN-α/β and

–γ only. (51, 62, 63)

11.6. Physiological effects of Interferons

IFNs play pivotal role in many of the biological phenomenon related to immunity, inflammation, and cell proliferation. IFNs are known as immune- inducer, immune-regulators, mediators and modulator of inflammation and inhibition of cell proliferation. IFNs show potent antiviral and antitumor activity, hence are used in viral disease. The IFN-α is widely used nowadays for viral hepatitis while IFN-β is used for immune mediated disease like multiple sclerosis (64). IFNs are proved anti tumor agents as reported previously. IFNs chemotherapy results in the tumor regression of a number of tumors including the; HL (Hodgkin’s lymphoma), NHL (non- Hodgkin’s lymphoma), CML

(Chronic myeloid leukemia), Hairy cell leukemia, Carcinoma of breast and kidneys, Kaposi`s carcinoma and malignant melanoma.

11.6.1. Interferons as immune modulators

IFNs contribute much in induction, modulation and regulation of immune reactions of body. The IFNs activate immune cells and increase secretion of other cytokines which in turn further stimulate the immune response. For example, the IFN-α stimulates Natural Killer (NK) cells which are the most potent cells of innate immunity having antibacterial, antiviral and antitumor activity.(65) IFN-α stimulates and activates the cellular immunity T cells marked by CD4+ and CD8+ markers. T cells are powerful cells of the acquired

55 immunity because they are cytotoxic. (66). Dendritic cells are unique professional antigen presenting cell which are stimulated also by IFN-α. (67)

Both the IFN-α and -γ induce the expression of HLA (Human leukocyte antigens). The HLA is also known as the MHC family which stands for the

Major histocompatibility complexes. The MHC has 2 classes - class I and

Class II MHC.

The MCH complex is crucial in producing acquired immunity by presenting antigens (68). Additionally, the IFN-α induces the expression of various cytokine types which are involved in the regulation of the immune responses. IFN-α up regulates interleukin-15 which stimulates CD8+ T-cells and Natural Killer cells (69). The IFN-α stimulated NK cells in turn causes increases secretion of IFN-γ level, which is essential for monocyte- macrophase system and T cells also (70). Clinically, the production of the IFN-

α is linked with different auto-immune diseases such as the IDDM (insulin- dependent diabetes mellitus) (71), RA (rheumatoid arthritis) (72), psoriasis

(73), systemic lupus erythematosus (74), and Sjögren’s syndrome (75). 11.6.2. Interferons as anti-proliferative agents

The IFNs exert anti-proliferative effects through their ability of inducing apoptosis. The apoptosis is a programmed cell death mediated through DNA translation and other molecular events. IFNs-mediated apoptosis in tumor cells have been reported to be produced by both IFNs type I and type II.

IFN-induced apoptosis is mediated through activation of both intrinsic

(mitochondrial) and extrinsic pathways. In particular, IFN-α mediated apoptosis is mediated through;

 Fas expression (extrinsic pathway) mediated apoptosis is reported in

basal cell carcinoma, leukemia and malignant melanoma.(76, 77)

 IFN-α also cause’s release of cytochrome c, activation of capsase-9, which

in turn stimulates the downstream “caspases” like the caspase-3, 25

caspase-6, and caspase-7. All of these caspases are involved in the

intrinsic or mitochondrial pathway of apoptosis (78).

11.6.3. Interferons as inflammatory modulators

The IFN-α/β and IFN-γ inhibit synthesis and secretion ofinterleukin-1

(IL-1) and prostaglandin (PG), both of which are important mediators of inflammation. The IL-1 and PG cause fever, hypotension and other features of inflammation.(79)Thus IFN-α, -β and -γ may be used for alleviating symptoms of inflammation, particularly in IL-1 mediated diseases.(80)

The IFN-α therapy augments synthesis and secretion of TNF-α. Thus

TNF-αmay be used as marker inflammation mediated by IFN-α (81)

11.6.4. Side effects of IFN (interferon) therapy

IFNs have been used as potent anti-viral, anti-tumor, and immuno- modulating agents successfully, but still it carries a significant risk. (82)

The symptoms reported with IFN therapy include depression, autoimmune reactions, and flu like symptoms. (83 - 85) Other studies have reported autoimmune reactions against red blood cells, platelets and coagulation factors resulting in autoimmune hemolytic anemia, autoimmune

57 thrombocytopenia, and abnormal clotting. Abnormalities of blood clotting and the hemolytic anemia are noted in 50% of the patients (86 - 88)

11.6.5. Recombinant Human IFN- α2a

IFNs have been permitted for use in humans to treat viral infections and cancer. Human recombinant IFN-α2a (Roferon®-A) and IFN-α2b (Intron® A).

Roferon-A® and Intron®A in humans are approved for the treatment of Hairy

Cell Leukemia, Malignant Melanoma, Follicular Lymphoma, Condylomata

Acuminata, AIDS-related Kaposi’s sarcoma, and Chronic Hepatitis C and B, in

Europe and USA (89). They are administered through intramuscular, subcutaneous and intralesional injections, and topical applications. IFN-β has been approved to treat relapsing multiple sclerosis in US (66, 68, 90).

CHAPTER III

MATERIALS AND METHODS

1. STUDY DESIGN

 An Experimental-Interventional Study

2. STUDY SETTING

 Animal House, Sindh Agriculture University Tando Jam, Isra University

Hyderabad and Diagnostic & Research Laboratory LUMHS

Hyderabad/Jamshoro

3. DURATION OF STUDY

 3 years (afterapproval of synopsis) 4. SAMPLE SIZE

 Eighty albino adult Wistar male rats (N=80) 5. ANIMAL GROUPS:

 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)

6. SAMPLING TECHNIQUE

Non-probability (purposive) sampling.

7. SAMPLE SELECTION:

Albino Wistar male rats were selected in a systemic way according to

inclusion and exclusion criteria:

7.1 Inclusion Criteria

 Adult albino male rats only

 Rats weighing 200-250 grams

7.2 Exclusion Criteria  Sick rats  Rats not feeding well  Moribund rats

8. ETHICAL COMMITTEE APPROVAL

 Institute ethical clearance

The research proposal was submitted to ethical review committee (ERC) of the

Isra University for conducting the research. The study protocol was approved by ERC.

 Animal ethical clearance

Animal “Ethical Clearance” was taken from the Institute ERC of the Faculty of

“Animal Husbandry and Veterinary Sciences”, Sindh Agriculture University,

Tando Jam. The animals were handled according to the NIH Guidelines for the

Care and Use of Laboratory Animals.

9. ANIMAL PROTOCOL &HOUSING

Albino Wistar male rats (n=80) were obtained from the animal house of the “Animal Husbandry and Veterinary Sciences”, Sindh Agriculture University,

Tando Jam. The animals were housed and handled according to the NIH

Guide for the Care and Use of Laboratory Animals.

The rats were kept in stainless steel cages. The cages were equipped with stainless steel feed containers and plastic drinkers with stainless nozzles.

The animals were kept in cage under a hygienic and well ventilated environment. Rats were fed lab chow feed. The tap water was given ad libitum. The light/dark cycle was maintained on 12 h intervals. All animal procedures were conducted under an animal protocol approved by Sindh

Agriculture University, Tando Jam. The cages of rats of controls and experimental groups were labeled as showing different parameters.

10. EXPERIMENTAL DETAILS

The control group was given normal saline injections, whereas the experimental groups received the recombinant human interferon-α-2b.

Interferon was kept in standard ice boxes at recommended temperature in deep freezers. Cold chain was strictly maintained from place of purchase to the experimental laboratory.

The recombinant human interferon-α-2b was administered as intraperitoneal injections thrice a week for three weeks. Animals were left for one

61 week more. At 30th day of post-interferon injections, all rats were given anesthesia.

 Animals were anesthetized by Ketamine (10 mg/Kg)and Xylazine

(0.5 mg/Kg)

 Cardiac puncture was performed for the collection of blood

samples within glass tubes containing in EDTA and also in the

Plain tubes as per standard protocol of sterilization.

o Blood sera were used for hormonal assay

 Animals were sacrificed by cervical dislocation

 Sperm were isolated by 20 gauge needle attached Disposable

syringe from Epididymis

o Smears were prepared and stained with Leishman`s and

Hematoxylin & Eosin staining for microscopy

 Orchidectomy was performed

One testicular sample was stored in marked containers in 10% formaldehyde as preservative/ fixative for Hematoxylin and Eosin staining and histology. Other testicular samples were kept at cold containers for genomic studies until testes were stored in deep freezers at -70oC.

11. HISTOLOGY AND PHOTOGRAPHY

The manual procedure was adopted to process the formaldehyde fixed tissue. The slides thus obtained wereobserved and studied under microscope.

Macro & Microscopic photography were carried out.

- Measurements were carried out by perimetry (stage micrometry 0.1)

Thomas Scientific Catalog No. 6585E65.

12. SPERM MORPHOLOGY

Direct smears were prepared from semen specimen, air dried and then stained with Leishman`s and Hematoxylin and Eosin for light microscopy.

13. TESTOSTERONE ASSAYS

Preparation of Assay-Specific Reagents (Cayman Chemicals, USA)

Testosterone EIA Standard

Testosterone EIA standard (Cayman Chemicals, USA) was prepared using the equilibrated pipette tip. Testosterone EIA Standard (100 μl) was transferred into a sterilized test glass tube. It was diluted with 900 μl ultrapure

H2O. This produced a concentration of 5ng/ml of this bulk standard solution.

EIA standard was prepared by selecting 8 test tubes labeled as 1-8. EIA Buffer

(900 μl) was aloqeted to test tube No. 1 and 500 μl to test tubes No. 2 to 8.

Approximately 5 ng/ml of standard solution was shifted to the tube-1 and was mixed continuously. Then the standard solution was diluted and concomitantly

500 μl was removed from the tube-1. Then it was placed in the test tube-2 and again mixed in same way. Again 500 μl solution was taken from test tube-2 and shifted to the test tube-3 and was mixed in the same way. This was

63 repeated similarly for tubes numbers 4-8. Diluted standards if not used within

24 hours, were discarded.

Testosterone AChE Tracer

Testosterone AChE Tracer was reconstituted as;

Testosterone AChE Tracer (100 dtn)was reconstituted with 6 ml “EIA

Buffer”and was stored at 4°Cfor to be used within 4 weeks.

Testosterone EIA Antiserum

Testosterone EIA Anti-serum was reconstituted as;

Testosterone EIA Antiserum (100 dtn) was reconstituted with 6 ml EIA

Buffer and stored at 4°Cand was used within 4 weeks.

Performing the Assay

Pipetting

 Different tips were used for Pipetting reagent

 Each reagent was equilibrated slowly

 Never exposed pipette tip to a reagent in the well

Addition of the Reagents

1. EIA Buffer

 100 μl EIA Buffer was added to the NSB wells.

 50 μl EIA Buffer was added to the B0 wells.

 50 μl of culture medium was substituted EIA Buffer

 50 μl of culture medium was added to the NSB and B0 wells

2. Testosterone EIA Standard

 50 μl was added from tube 8 to both of lowest standard wells

 50 μl was added from tube 7 to next of two standard wells

 Procedure was continued until standards were aliquoted

 Pipette tip was used to aliquot the standard tubes

 Pipette each standard to equilibrate

3. Samples

 50 μl of the sample poured into each of the well,

 This was assayed at 2 dilutions which was minimal requirement

 The dilutions were performed in duplicate times

4. Testosterone Ach E Tracer

 50 μl of tracer was poured into each well

5. Testosterone EIA Antiserum

 50 μl of anti serum was added to each well.

Incubation of the Plate

 Each plate was covered with a plastic film. It was incubated for 2 hours on

an “orbital-shaker” at the room temperature.

Plate development

 “Ellman’s Reagent” was reconstituted before use

 20 ml of reagent was used as it was sufficient to produce 100 well

 100 dtn vial “Ellman’s Reagent” was reconstituted with 20 ml of Ultra Pure

H20.

 Each of the well was emptied. Then rinsed five time with the help of buffer-

wash

 Then approximately 200 μl “Ellman’s Reagent” poured into the well

 Now 5 μl of “Tracer” was added each of the TA wells

 Plates were covered with thin “plastic film”

 Orbital-shaker was used to develop optimal plates

 Assay was developed in 60-90 minutes (i.e., B0 wells ≥0.3 A.U. (blank

subtracted).

Standard curve

Standard curve Reading the Plate

1. Plate bottom was wiped with clean tissue for removing fingerprints, etc

2. Plate cover was removed, care was taken to prevent splashing of

“Ellman’s-Reagent”

3. Readings were taken at the 405 - 420 nm wavelength

4. Absorbance was noted serially until B0 wells reached a minimum of 0.3

A.U.

5. Plate was read at the absorbance range of 0.3-1.0 A.U (B0 wells)

14. SERUM FSH ASSAY

Performing the Assay (Cayman Chemicals, USA)

Pipetting

• Tip of pipettes were equilibrated for each of the reagent, before it was begun

• Tips of the pipette were not allowed to be exposed to the reagent(s) already present in the well.

Addition of the Reagents

1. FSH Standards

Each standard (50 μl) was added to appropriate wells.

2. Samples

Sample (50 μl) was added to appropriate wells.

3. Anti-FSH-HRP + Anti-FSH-Biotin Conjugate

Antibody mixture (100 μl) was added to each well, except the Blk wells.

Incubation of the Plate

The plate was covered with the plastic film and incubated at room temperature

(22°C-28°C) for one hour.

Development of the Plate

1. The wells were emptied. They were washed 3x times with diluted Wash

Buffer. Each well was completely filled with Wash Buffer during each wash.

The plate was inverted between wash steps to empty the fluid from the wells.

The inverted plate was gently tapped on the absorbent paper in order to remove the residuals of the “Washed buffer”, finally.

2. TMB Substrate Solution (100 μl) was added to each well of the plate, including the Blk wells.

3. Incubated for exactly 15 minutes at room temperature in the dark.

4. Stock Solution (100 μl) was added to all wells and in the same order and same rate as the addition of TMB Substrate in Step 2.

Standard curve

Reading the Plate

14. A clean tissue paper was used to wipe the bottom of plate to omit the

finger prints , dirt, etc

2. The plate was read at a wavelength of 450 nm.

3. The optical density (O.D.) of standard 6 should be ≥1.3.

15. SERUM LH ASSAY

Performing the Assay (Cayman Chemicals, USA)

Addition of the Reagents

1. Luteinizing Hormone Standards

20 μl of each standard was added to appropriate wells.

2. Samples

20 μl of sample was added to appropriate wells.

3. Anti-LH Conjugate

100 μl conjugate was added to each well (except the Blk wells).

Incubation of the Plate

The plate was covered with the plastic film and incubated at room temperature

(22°C-28°C) for one hour.

Development of the Plate

1. Empty the wells and wash twice with diluted Wash Buffer. Each well should be completely filled with Wash Buffer during each wash. Invert the plate between wash steps to empty the fluid from the wells. The plate was inverted between wash steps to empty the fluid from the wells. The inverted plate was gently tapped on the absorbent paper in order to remove the residuals of the

“Washed buffer”, finally.

2. 100 μl of TMB Substrate Solution was added into each well of the plate, including the Blk wells.

3. And were incubated for 15 minutes (exactly) at room temperature in the dark.

4. Stock solution (100 μl) was added to all wells and in the same order and same rate as the addition of TMB Substrate in Step 2.

Standard curve

Reading the Plate

1. A clean tissue paper was used to wipe the bottom of plate to omit the finger

prints , dirt, etc

2. The plate was read at a wavelength of 450 nm.

3. The optical density (O.D.) of standard 6 should be ≥1.3. 16. AROMATASE GENE DETECTION Total RNA extraction from testicular tissue:

The total RNA was taken out from the testicular tissue of the experimental rats with RNA Extraction commercial Kit (Tissue Total RNA Kit,

Favorgen Bioteck Corp. Cat. #: FATRK-001) according to manufacturer’s guidelines. Briefly; the frozen tissue sample (30mg) was cut into small part with scissors and transferred into centrifuge tube then grinded with sterilized micropestle to breakup in small pieces, 350μl of FARB Buffer (ß-ME added) was added to the grinded tissue sample. The tissue sample sheared by passing lysate 10 times through a 20-G needle syringe and incubated at room temperature for 5 min. A Filter Column was placed into a Collection Tube and sample mixture was transferred to Filter Column, then centrifuged at “14,000” rpm for approximately two minutes. Clear supernatant was shifted from the collection tube to a new autoclaved micro centrifuge tube and adjusted the volume of the clear lysate then same volume of 70% Ethanol was added to the clear lysate and mixed well by vortexing, tube was briefly spin to remove drops from the inside of the lid. A FARB Mini Column was placed to a Collection

Tube and transferred the ethanol added sample mixture (including any precipitate) to the FARB Mini Column. Centrifuged at 14000 rpm for 1 min,

73 discarded the flow-through and FARB Mini Column was returned back to the

Collection Tube then 500 µl of Wash Buffer 1 was added to the FARB Mini

Column, then centrifuged at “14000” rpm for one minute. The flow-through was discarded and returned the FARB Mini Column back to the Collection Tube and 750µl of Wash Buffer 2 (Ethanol Added) was added to the FARB Mini

Column, centrifuged at full speed (14000rpm) for 1 min. The flow-through was discarded and returned the FARB Mini Column back to the Collection Tube.

After one more washing, again 750 µl of Wash Buffer 2 (Ethanol Added) was added to the FARB Mini Column, centrifuged at full speed (14000rpm) for 1 min. The flow-through was discarded and returned the FARB Mini Column back to the Collection Tube. The FARB Mini Column, it was centrifuged at

14000rpm for an additional 3 minutes to dry. The FARB Mini Column was placed to a Elution Tube (1.5 ml micro centrifuge tube) and 50 µl of RNase- free ddH2O was added to the membrane center of the FARB Mini Column then Mini Column was left to stand for 1 min. The FARB Mini Column was centrifuged at full speed (14000rpm) for 1 min to elute RNA. After this stage, samples were stored in ice until quantification then stored at -700C.

Quantification of Total RNA

Total RNA concentration present in the samples was quantified with a

Nanodrop spectrophotometer (Nanodrop Technologies, USA). Absorbance readings were taken at 260 nm and 280 nm. Standard procedure was adopted. Briefly; the spectrophotometer was first blanked with distilled water alone and extent was made on distilled water. The measurement at 260 nm made available the quantity of nucleic acid (RNA). The ratio of the 260 / 280

74 measurements were considered for the purity of the sample, for RNA a ratio between 1.8 – 2 was considered good to obtain accurate results. All samples were measured at 260nm to calculate the quantity of total RNA present within the ratio of 1.8-2.

Gene expression analysis

The Gene expression analysis was done through the Applied Biosystem

7300 (Foster city, CA, U.S.A) through the TOPreal™ One-step RT qPCR Kit

(SYBR Green with high ROX) according the manufacturer’s instruction. Briefly,

20 μl RT qPCR Mixture was assembled on ice as;

TOPreal™ One-step RT qPCR Enzyme 1 μl

MIX

2X TOPreal™ One-step RT qPCR 10 μl

Reaction MIX

Template (total RNA, 10 ng ~ 1 μg/μl) 1 μl

Primers 1 (5~10 pmol/μl) 1 μl

Primers 2 (5~10 pmol/μl) 1 μl

Sterile water (RNase free) 6 μl

RT qPCR cycle condition was adopted as;

Reverse Transcription 50 30 min qPCR Condition with 40 cycles℃

Initial denaturation 95 10 min

Denaturation 95℃ 5 Sec

Annealing & Elongation 60℃ 30 Sec

℃

RT qPCR was done by using the primers, Aromatase (Sense): 5’-

CCACTAAGGGC AAGATGAT-3, Aromatase (Anti-sense):

5’GGGTTCAGCATTTCCAAAAA-3’, β -actin (Sense): 5’-TCGTG

CGTGACATTTGAG-3 and β -actin (Anti-Sense): 5’-ATTGCCGAT

AGTGATGACCT-3’. β Actin was utilized as a internal control for normalization of quantification of Aromatase. Quantification of the results was done

ΔΔCT(delta Ct method) method of relative quantification with StepOne

Software and StepOne Plus Real-Time PCR system (Applied Biosystem).The averageCT was measured for both Aromatase andβActinwhereas using the formula CT = (CT, Aromatase - CT, β Actin ) , the difference in the Ct was resolute. Delta Ct method or CT method was applied for the comparative expression of Aromatase. Furthermore, values obtained through CT level using real-time PCR machine were shifted to a spreadsheet program such as

Microsoft Excel. The change in expression of the Aromatase gene was standardized to β-actin. Also fold change in the Aromatase gene regulated through β -actin and relative expression of Aromatase was measured for each sample accordingly.

Statistical analysis:

The results obtained were presented as mean ± standard deviation (SD).

Aromatase mRNA relative expression was done by one-way ANOVA in SPSS

22.0 for windows statistical package (SPSS Inc., Chicago, IL, USA). The level of significance were kept at P <0.05.

Result

Aromatase mRNA Expression of testicular sample

In the biotechnology, Real-time PCR is a trustworthy device for the measurement and quantification of mRNA of any desirable gene at transcription level and that has been associated with provision of useful assessment for the gene appearance. Endogenous control gene that is also called housekeeping genes are made available to normalize mRNA levels between samples for sensitive comparisons of mRNA transcription. Known primers for both Aromatase and β Actin were used for amplification process during RT PCR. Quantitative PCR analysis indicated that Aromatase showed a dose dependent expression in the testicular tissue with increase in the concentration of interferon-α-2b in the treated groups as indicated in Figure.

The finding of the current study indicated that The Aromatase mRNA expression showed a significant decrease in 10 MIU treated group in comparison with control and other treated groups. It is first research investigation to elaborate changes of Aromatase mRNA expression in the testicular tissue of the experimental albino rat with different concentration of the interferon-α-2b .Thus the present study demonstrated the down regulation of Aromatase at high concentration of the interferon-α-2b. Further study would be needed to clarify the Aromatase protein synthesis and its regulatory mechanism by the testicular tissue with different concentration of interferon-α

(91, 92).

17. PREPARATION OF HISTOLOGICAL STUDIES:

DISSECTION OF RAT:

Materials required:

i. Freshly killed rat

ii. Dissecting pan with wax bottom

iii. Dissecting box

iv. Dissecting pins

PROCEDURE:

Skinning the Ventral Surface:

The rat was fixed in the dismembering skillet with ventral surface upward, by sticking the four appendages to the base of dissecting dish.

Opening of Abdominal wall: i- Incision was made along the abdominal wall ventrally with the help of scissors pair. Forceps were used for muscle holding to avoid tissue and viscera damage. ii- Incision was made along midline, beginning upward to the ribs and downward ending close to genitalia. iii-Two parallel entry points were made each on right and left side close to the base of rib pen. These cuts were developed upto the stuck back skin. iv-The folds of body wall were collapsed and stuck to the wax so the abdominal approach was utilized to reach to scrotum to evacuate the testes.

ANIMAL MATERIAL

We used grown-up Wistar albino rats of Jinnah Postgraduate Medical

Center strain. Specifically, Charles River Breeding Laboratories, Brooklyn,

Massachusetts, U.S.A. Wistar rats were cross reproduced at Animal House of

Basic Medical Sciences Institute (BMSI), Jinnah Postgraduate Medical Center

(JPMC) Karachi. The animals were on research diet. One week prior to commencement of study, the animals around 200±5 grams weight were examined for physical well-being. Ninety (90) animalswere then chosen according to this criterion, and eventually, were chosen for the trial study. Eventually, the study was performed on a net number of 80 albino rats.

SACRIFICE OF ANIMALS:

Every one of the animals were continued fasting overnight preceding penance at 24 hours treatment, separately. Animals were anesthetized by

Ketamine (10 mg/Kg) and Xylazine (0.5 mg/Kg). They were set on adissection board and their mid abdominal region was opened with a long midline cut. Testes were recognized and their gross appearance was noted for any undeniable change with lens and were expelled and dried by tissue paper. The tissues were quickly exchanged to typical saline for histopathological examination.

TISSUE PRESERVATION:

Testes were immersed in normal saline for 24-48 hour, dried out in rising evaluations of alcohol, cleared in xylene and embedded in parables. 3µ thick longitudinal and transverse areas were cut on microtome, drifted in high temperature H2O at 42°C and mounted on altogether clean glass slides fittingly numbered with a pencil.

The slides were set on hot plate at 37°C for 24 hours for fixation of tissue sections. Hematoxylin and Eosin (H & E) staining was applied.

Data analysis

Data were analyzed using SPSS version 21.0 (Chicago, Illinois, USA) for windows release. Normality of data distribution was analyzed by Shapiro Wilks.

The continuous variables were analyzed by ANOVA and results were presented as mean ± SD. The categorical variables were analyzed by Chi-square test and results were presented as frequency and percentages.Data was presented in tables, graphs and charts.Microsoft excel was also used for graphing. A p-value of

≤ 0.05 was considered statistically significant.

CHAPTER IV

RESULTS

The study variables serum testosterone, FSH and LH,morphometricand histomorphometric variables were analyzed by analysis of variance, descriptive statistics, post Hoc Fischer` LSD test, and chi square test, respectively. The study variables which showed significant F-ratio and p-value were applied further for post Hoc test.

Analysis of varianceshowed significant F-ratio and p-values for all research variables as depicted in table IV-1.

 Serum testosterone

Serum testosterone reduced progressively in interferon treated animals at different doses compared to controls. Serum testosterone, mean± SD, in control groups was 0.81±0.04versus0.74±0.03, 0.67±0.04 and 0.55±0.6 pg/mL in groups

II, III and IV respectively (p≤ 0.011) (table IV-2 and graph IV-1).

 Serum follicle stimulating hormone (FSH)

Serum FSH reduced progressively in interferon treated animals at different doses compared to controls. Serum FSH, mean± SD, in control groups was 7.43±

0.79compared to6.6±0.75, 6.02±0.82 and 5.2±1.18 mIU/ml respectively (p≤ 0.026)

(table IV-3 and graph IV-2).

 Serum luteinizing hormone (LH)

Serum LH also showed a reduction in interferon treated animals at different doses compared to controls.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 mIU/ml respectively

(p≤0.003) (table IV-4 and graph IV-3).

 Histomorphometric measurements

Findings of histology and histomorphometric parameters of seminiferous tubules are shown in table IV-5 to IV-14(Graphs IV-4 to IV-13) respectively.

 Thickness of seminiferous tubules

Seminiferous tubules were thin in experimental rats compared to controls.

Thickness of seminiferous tubules in controls, rhINF-3MIU,rhINF-5MIU andrhINF-

10MIUwas noted as 195.55±5.40, 181.20±7.82, 169.20± 2.35 and 155.90± 7.08

µm respectively. Table IV-5 and graph IV-4 shows the details of thickness of seminiferous tubules.

 Diameter of seminiferous tubule

Diameter of seminiferous tubule was increased in experimental rats.

Diameter of seminiferous tubules in controls, rhINF-3MIU,rhINF-5MIU andrhINF-

10MIU was noted as 9.37±2.09, 14.21±3.17, 16.97± 3.79 and 15.22± 3.40 µm respectively. Table IV-6 and graph IV-5 shows the calculated values of diameter in different rat groups.

 Seminiferous tubule length

Seminiferous tubule length was reduced in experimental rats compared to controls. Length of seminiferous tubules in controls, rhINF-3MIU,rhINF-5MIU andrhINF-10MIU was noted as 13.52± 0.81, 12.86± 0.80, 12.08± 0.62 and 11.28±

0.62 µm respectively. Table IV-7 and graph IV-6 shows the details of length of seminiferous tubules.

 Germ cells counts

Germ cell counts were reduced in experimental rats compared to controls.

Germ cell counts in controls, rhINF-3MIU, rhINF-5MIU and rhINF-10MIU were reduced in 2 (10%), 11 (55%), 17 (85%) and 19 (95%) rats respectively compared to control. Table IV-8 and graph IV-7 shows the germ cell counts.

 Germ cell maturation

Germ cell maturation wasarrested in experimental rats compared to controls. Germ cell maturation arrest in controls, rhINF-3MIU, rhINF-5MIU and rhINF-10MIU were noted in 0 (%), 3 (15%), 16(80%) and 19 (95%) rats respectively. Table IV-9 and graph IV-8 shows normal, reduced and arrested maturation of germ cells.

 Desquamation

Seminiferous tubule desquamation was noted as in 0 (0%), 9 (45%),

2(10%) and 3(15%) rats in controls and experimental groups respectively.Desquamationwas observed prominently in experimental rats of rhINF-3MIU group.Table IV-10 and graph IV-9 shows positive and negative counts of desquamation in various rat groups.

 Basement membrane (BM)

Thick BM was found in more numbers of experimental rats compared to controls as shown in Table IV-11 and graph IV-10. Basement membrane thickness was noted in 1(5%) in controls and 6(30%), 15 (75%) and 17(85%) experimental rats respectively.Increased thickness of BM was prominent in the high dose rhIFN-10MIU treated rats.

 Interstitial edema

Interstitial edema was noticeably noted in the experimental rats compared to controls as shown in Table IV-12and graph IV-11. 1 (5%) of controls showed

83 interstitial edema, compared to8 (40%), 16 (80%) and 18 (90%) in experimental groups II, III and IV respectively.Interstitial edema was noticeable finding in high dose rhIFN-10MIU treated rats.

 Vascularity

Hypervascularity was a prominent feature of the rhIFN treated rats on histopathological examination. Hypervascularity was noted in 2 (10%), 4 (20%),

15(75%) and 17 (85%) rats respectively.

 Sertoli cell counts

Increased Sertoli cell counts were noted in the rhIFN treated experimental rats as shown in Table IV-13 and graph IV-12. Increased Sertoli cell counts in controls, rhINF-3MIU, rhINF-5MIU and rhINF-10MIU were noted as in 0 (0%), 13

(65%), 16 (80%) and 18 (90%) rats respectively.

 Leydig cell counts

Increased Leydig cell counts were noted in the rhIFN treated experimental rats compared to controls as shown in Table IV-14 and graph IV-13. Increased

Leydig cell counts in controls, rhINF-3MIU, rhINF-5MIU and rhINF-10MIU were noted in 0 (0%), 12 (60%), 16 (80%) and 19 (95%) respectively.

Table IV-1. Analysis of variance showing F-ration and p-value of various study parameters in 80 rat models distributed into four equal groups Sum of df Mean F p-value Squares Square Between 0.72 3 0.24 113.24 Groups 0.0001 Serum Within 0.16 76 0.002 testosterone Groups (pg/mL) 0.88 79 Total Between 53.01 3 17.67 21.48 Groups 0.0001 Serum FSH Within 62.52 76 0.82 (mIU/mL) Groups 115.54 79 Total Between 92.46 3 30.82 35.50 Groups 0.0001 Serum LH Within 65.98 76 0.86 (mIU/mL) Groups 158.45 79 Total Between 17166.73 3 5722.24 156.59 Groups 0.0001 Thickness of ST Within 2777.15 76 36.54 (TST) (µm) Groups 19943.88 79 Total Between 5584.15 3 1861.38 9.19 Groups 0.0001 Diameter of ST Within 15389.40 76 202.49 (DST) (µm) Groups 20973.55 79 Total Between 56.52 3 18.84 36.19 Groups 0.0001 Total tubular Within 39.56 76 0.52 length (m) Groups Total 96.08 79

Table IV.2. Serum testosterone (pg/mL) 95% Mean SD Confidence Minim Maxim p- Groups Interval for um um value

Mean Lowe Upper r Bound Boun d

0.81 0.04 0.79 0.83 0.75 0.89 Group I- Control (n=20)

≤

Group II- rhIFN - 3MIU 0.74 0.03 0.72 0.75 0.69 0.79 0.011

(n=20)

Group III. rhIFN 5 MIU 0.67 0.04 0.65 0.69 0.61 0.78

(n=20)

Group IV. rhIFN- 10MIU 0.55 0.06 0.52 0.58 0.49 0.73

(n=20)

Table IV.3. Serum FSH (mIU/mL) 95% Mean SD Confidence Minim Maxim p- Groups Interval for um um value

Mean Lowe Uppe r r Boun Boun d d

Group I- Control 7.43 0.79 7.05 7.80 5.65 8.79

(n=20)

≤ Group II- rhIFN - 3MIU 6.67 0.75 6.32 7.03 5.14 7.69 0.026

(n=20)

Group III. rhIFN 5 MIU 6.02 0.82 5.64 6.41 3.98 7.13

(n=20)

Group IV. rhIFN- 5.22 1.18 4.66 5.77 3.12 7.12

10MIU (n=20)

Table IV.4. Serum LH (mIU/mL) 95% Mean SD Confidence Minimum Maximum p- Groups Interval for value

Mean Lower Upper Bound Bound

Group I- 7.56 0.70 7.23 7.89 6.62 8.79

Control (n=20)

6.83 0.63 6.53 7.12 5.43 7.69 Group II- rhIFN ≤

- 3MIU (n=20) 0.003

Group III. 5.65 0.92 5.22 6.08 3.98 7.10 rhIFN 5 MIU

(n=20)

Group IV. 4.76 1.31 4.14 5.37 2.23 7.12 rhIFN- 10MIU

(n=20)

Table IV.5. Thickness of seminiferous tubule (µm) 95% Mean SD Confidence Minimum Maximum p- Groups Interval for value

Mean Lower Upper Bound Bound

Group I- 195.55 5.40 193.02 198.07 178.00 203.00

Control

(n=20) ≤ 0.0001 Group II- 181.20 7.82 177.53 184.86 165.00 192.00 rhIFN -

3MIU (n=20)

Group III. 169.20 2.35 168.09 170.30 164.00 173.00 rhIFN 5 MIU

(n=20)

Group IV. 155.90 7.08 152.58 159.21 141.00 168.00 rhIFN-

10MIU

(n=20)

Table IV.6. Diameter of seminiferous tubule (µm) 95% Confidence Mean SD Interval for Mean Minimum Maximum p- Groups value

Lower Upper Bound Bound

Group I- 9.37 2.09 273.6854 255.00 280.00 9.37017

Control

(n=20) ≤ 0.005 Group II- 14.21 3.17 271.5550 245.00 287.00 14.21970 rhIFN - 3MIU

(n=20)

Group III. 16.97 3.79 259.7448 230.00 277.00 16.97552 rhIFN 5MIU

(n=20)

15.22 3.40 256.8255 228.00 277.00 15.22498 Group IV. rhIFN- 10MIU

(n=20)

Table IV.7. Seminiferous tubules length (m) 95% Mean SD Confidence Minimum Maximum p- Groups Interval for value

Mean Lower Upper Bound Bound

Group I- 13.52 0.81 13.14 13.91 11.98 14.78

Control (n=20)

≤ 12.86 0.80 12.48 13.23 11.56 13.71 0.001 Group II- rhIFN

- 3MIU (n=20)

12.08 0.62 11.79 12.37 11.20 13.15 Group III. rhIFN

5MUH (n=20)

Group IV. 11.28 0.62 10.99 11.57 10.54 12.56 rhIFN- 10MIU

(n=20)

Table IV-8. Germ cell counts in seminiferous tubules

2 Groups Normal Decreased X p-value

Group I- Control 18 02

(n=20)

36.81 0.001 Group II- rhIFN - 09 11

3MIU (n=20)

Group III. rhIFN 5 03 17

MIU (n=20)

Group IV. rhIFN- 01 19

10MIU (n=20)

Table IV -9. Germ cell maturation

2 Groups Normal Decreased Arrested X p-value

Group I- Control 19 01 0

(n=20)

95.96 0.0001 Group II- rhIFN - 03 14 03

3MIU (n=20)

Group III. rhIFN 5 0 04 16

MIU (n=20)

Group IV. rhIFN- 0 01 19

10MIU (n=20)

Table IV- 10. Seminiferous tubule desquamation

2 Groups Positive Negative X p-value

Group I- Control 0 20

(n=20)

14.68 0.02 Group II- rhIFN - 09 11

3MIU (n=20)

Group III. rhIFN 5 02 18

MIU (n=20)

Group IV. rhIFN- 03 17

10MIU (n=20)

Table IV-11. Seminiferous tubule basement membrane

2 Groups Normal Thick X p-value

Group I- Control 19 01

(n=20)

34.17 0.001 Group II- rhIFN - 14 06

3MIU (n=20)

Group III. rhIFN 5 05 15

MIU (n=20)

Group IV. rhIFN- 03 17

10MIU (n=20)

Table IV-12. Interstitial tissue

2 Groups Normal Edematous X p-value

Group I- Control 19 01

(n=20)

36.75 0.0001 Group II- rhIFN - 12 08

3MIU (n=20)

Group III. rhIFN 5 04 16

MIU (n=20)

Group IV. rhIFN- 02 18

10MIU (n=20)

Table IV -13. Sertoli cell counts

2 Groups Normal Decreased Increased X p-value

Group I- Control 17 03 0

(n=20)

48.45 0.0001 Group II- rhIFN - 03 04 13

3MIU (n=20)

Group III. rhIFN 5 02 02 16

MIU (n=20)

Group IV. rhIFN- 01 01 18

10MIU (n=20)

Table IV -14. Leydig cell counts

2 Groups Normal Decreased Increased X p-value

Group I- Control 15 05 0

(n=20)

47.71 0.0001 Group II- rhIFN - 04 04 12

3MIU (n=20)

Group III. rhIFN 5 01 03 16

MIU (n=20)

Group IV. rhIFN- 01 0 19

10MIU (n=20)

Graph IV-1. Serum testosterone in different rat groups

Graph IV-2. Serum follicle stimulating hormone in different rat groups

100

Graph IV-3. Serum luteinizing hormone in different rat groups

101

Graph IV-4.Thickness of seminiferous tubule in different rat groups

102

Graph IV-5.Diameter of seminiferous tubules in different rat groups

103

Graph IV-6Total seminiferous tubular length in different rat groups

104

Graph IV-7.Germ cell counts in different rat groups

105

Graph IV-8.Germ cell maturation in different rat groups

106

Graph IV-9.Seminiferous tubule desquamation in different rat groups

107

Graph IV-10.Seminiferous tubule basement membrane in different rat groups

108

Graph IV-11.Interstitial edema in different rat groups

109

Graph IV-12.Germ cell counts in different rat groups

110

Graph IV-13.Germ cell counts in different rat groups

111

 CYP-450-Aromatase (CYP- 19) mRNA gene studies

Testicular blended germ cell arrangement was acquired by the trypsin-

DNase strategy with a few adjustments. Quickly, after enzymatic processing, the cell suspension was washed in PBS supplemented with 3•3 mM glucose and 6 mM pyruvate (Sigma).

The subsequent suspension was separated first through fine nylon lattice to expel Sertoli cell totals, and after that through glass fleece to minimize spermatozoa content in the arrangement. Further detachment of testicular cell sorts was expert by unit gravity sedimentation through a BSA (Roche, Mannheim,

Germany) slope (0•2– 2•75%) in a Sta-Put apparatus.

PS-and RS-containing divisions were distinguished under a stage contrast microscope, and the most noteworthy homogeneity portions were pooled and washed no less than three times with PBS. These germ cells had a virtue more prominent than 95% as appeared by the nonappearance of physical cell sullying when inspected infinitesimally (morphological portrayal) and evaluated by other criteria, for example, histochemical recoloring and RT-PCR. 3β-Hydroxysteroid dehydrogenase (3β-HSD) histochemical recoloring, a particular marker of Leydig cells, was utilized to investigate the virtue of germ cell portions. To gauge the

Sertoli cell defilement of the germ cell arrangements a histochemical recoloring with Oil Red O (Chapin et al. 1987) was performed. Furthermore, a RT-PCR procedure was connected to open up undifferentiated cell variable (or Steel Cell

Factor, SCF), a known Sertoli cell marker and fibronectin (Fbr), a peritubular myoid cell item. Refined germ cells were in this manner utilized for brooding and microsomal extraction. The reasonability of germ cells previously, then after the fact hatching was assessed utilizing the trypan blue prohibition test (Sigma); the recolored cells were thought to be dead.

112

CYP450-Aroma (CYP-19) mRNA level and Aromatase activity

A “single band” was present in the seminiferous tubules after RT-PCR from the isolates of the purified germ cells. In present study, the positive control used was the rat ovary. A correct predicted size of 289 bp was observed from the amplified cDNA fragment using the CYP-450-Aroma specific primers. The immaculateness of germ cells was checked under a magnifying instrument after

Sta-Put utilizing variable number of testicular physical cell markers.Microscopic analysis showed that 2 germ cell fractions purities were >95% using morphological characterization.

Up to 5% of contaminants were spermatocytes of different stages. Few round spermatids and few elongated spermatids in the round spermatids fractions were observed. Germ cell preparations of present study were free from contamination of Sertoli cells as shown by Oil red O – a specific Sertoli cell coloration.

The specific transcripts for aromatase were fully translated as shown by

Aromatase activities noted as;

 88±21 fmol/mg protein/h from Pachytene spermatocytes

 157±10 fmol/mg protein/h from Round spermatids

113

Aromatase gene PCR

114

Ct (Cycle threshold) values of Gene expression

• In a real time PCR assay a positive reaction is detected by accumulation of a fluorescent signal.

• Ct is defined as the number of cycle threshold required for the fluorescent signal to cross the threshold.

• Ct values are inversely proportional to the amount of target nucleic acid in the sample

• Ct value Range:

– Ct ≤ 29 indicate strongly positive reaction, shows abundance of nucleic acid in the sample

– Ct 30 -37 indicate moderate positive reaction & moderate amounts of nucleic acid in the sample

– Ct >30-40 indicate weak positive reaction & indicates minimal amounts of nucleic acids in the sample

– Ct ≤ 29 indicate strongly positive reaction, shows abundance of nucleic acid in the sample

– Ct 30 -37 indicate moderate positive reaction & moderate amounts of nucleic acid in the sample

– Ct >30-40 indicate weak positive reaction & indicates minimal amounts of nucleic acids in the sample

115

TableTableVI -19.IV-15. Ct (cycleCt threshold threshold) levels values in controls in controls and and experimental experimental groups groups

Groups Mean SD Gene Expression p-value

Group I- Control (n=20) 23.99 3.99 Strong gene expression

Group II- rhIFN - 3MIU (n=20) 32.96 2.64 Moderate gene expression 0.0001 Group III. rhIFN 5 MIU (n=20) 36.42 2.08 Moderate gene expression

Group IV. rhIFN- 10MIU (n=20) 39.29 1.98 Mild/weak gene expression

ANOVA+ Tukey Cramer tests

116

Graph IV-14. Bar graphs showing Ct value of gene expression

117

 Histological examination

Texture studies of controls revealed that the seminiferous tubules were normal looking with intact basement membranes, epithelial cells layers, Sertoli cells and interstitial cells of Leydig. Normal vascularity was observed in seminiferous tubules.

On the other hand, the seminiferous tubules showed reduction in the lining epithelial cell layers andgerm cells. Interstitial cells of Leydig and Sertoli cells showed increased counts. Basement membrane showed increased thickness and defects in interferon treated animals.

Photomicrographs IV-1 to IV-14 show the histological findings of controls and interferon treated animals.

 Sperm morphology

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 as shown in Photomicrographs IV-15 to IV-35. 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.

118

GROUP I. CONTROL

Normal Normal Seminiferous Spermatoge Tubule nesis

Photo micrograph IV-1.Group I. Control- testicular tissue sections showing intact histological architecture, normal structure & normal spermatogenesis. (H & E stain x100)

119

GROUP I. CONTROL

Normal Normal Seminifero Spermatoge us Tubule nesis

Photomicrograph IV-2.Group I. Control- testicular tissue sections showing normal seminiferous tubules (ST) showing with intact histological architecture. (H &E stain x100)

120

GROUP I. CONTROL

Normal Normal Seminiferous Spermatog- Tubule enesis

Photomicrograph IV-3.Group I. Control- testicular tissue sections showing normal seminiferous tubules (ST) showing with intact histological architecture. (H &E stain x100)

121

Group II. rhIFN α-2b (3 mIU)

Maturation Arrest

Increase Decrease Sertoli Gems Cells Cells

Photomicrograph IV-4.Decrease germ cell, maturation arrest, increased numbers of Sertoli cells. (H & E x100)

122

Group II. rhIFN α-2b (3 mIU)

Thickened Spermato Basement genesis membrane Arrest

Photomicrograph IV-5.Thickened basement increased Leydig cell, spermatogenesis in arrest. (H & E stain x200)

123

Group II. rhIFN α-2b (3 mIU)

Increased Leydig Cells

Thickened Spermatoge Basement -nesis Arrest membrane

Photomicrograph IV-6.Thickened basement membrane, increased Leydig cell, spermatogenesis arrested. (H & E stain x200)

124

Group III. rhIFN- α-2b 5 mIU

Thick and Basement Membrane

Clumping Spermatoge of -nesis Arrest Epithelial

Cells

Photomicrograph IV-7.Decrease germ cells, maturation arrest, thickened basement membrane, prominent clumping of epithelial cells with relatively increased vascularity.(H & E stain x100)

125

Group III. rhIFN α-2b 5 mIU

Clumping of Increase Epithelia Sertoli Cells Cells

Maturation Decreased Arrest Germ Cells

Photomicrograph IV-8.Decrease germ cells, increased Sertoli cells, clumping of epithelial cells, thickened basement and arrest of sperm maturation. (H & E stain x100)

126

Group III. rhIFN- α-2b 5 mIU

Clumping of Increase Epithelia Sertoli Cells Cells

Maturation Decreased Arrest Germ Cells

Photomicrograph IV-9.Decrease germ cells, increased Sertoli cells, clumping of epithelial cells, thickened basement arrest of maturation, relatively increased length of tubules and vascularity. (H & E stain x100)

127

Group III. rhIFN- α-2b 5 mIU

Clumping of Increase Epithelial Sertoli Cells Cells

Maturation Increase Tubule Arrest Diameter Membrane

Photomicrograph IV-10.Thickening of basement membrane relatively increased in vascularity, spermatogenesis is arrested, decreased germ cell, increased Sertoli &Leydig cells, clumping of epithelial cells and increased the diameters of the tubules.(H & E stain x100)

128

Group IV. rhIFN- α-2b 10 mIU

Increase Increase Leydig Cells Sertoli Cells

Maturation Increased Arrest Vascularity

Photomicrograph IV-11.Increased length, diameter, width of tubules, spermatogenesis is arrest, increased number of Sertoli cells, increased number of Leydig cell, increased vascularity and thickened basement membrane.(H & E stain x100)

129

Group IV. rhIFN- α-2b 10 mIU

Increase Increase Vascularity Sertoli Cells

Maturation Decreased Arrest Germ Cells

Photomicrograph IV-12.Spermatogenesis is arrest, increased number of Sertoli cells, increased number of Leydig cell, increased vascularity increased, thickened basement membrane with increased diameter of the tubules.(H & E stain x100)

130

Group IV. rhIFN- α-2b 10 mIU

Increase Thickened Leydig Cells Basement & Membrane Vascularity

Maturation Increased Arrest & Diameter Increase of Tubules Sertoli Cells

Photomicrograph IV-13.Spermatogenesis is arrest, increased Sertoli cells, increased Leydig cell, increased vascularity and thickened basement membrane and increased the diameter of the tubules.(H & E stain x200)

131

Group IV. rhIFN- α-2b 10 mIU

Increase Thickened Leydig Cells Basement & Membrane Vascularity

Maturation Increased Diameter Arrest & Increase of Tubules Sertoli Cells

Photomicrograph IV-14.Spermatogenesis is arrest, increased the number of Sertoli cells, increased the number of Leydig cell, and increased vascularity, thickened basement membrane.(H & E stain x200)

132

Group I. Control

Normal Sperm Morphology & Count

Photomicrograph IV-15. Control showing normal sperm morphology Leishman’s staining on (x100)

133

Group I. Control

Normal Sperm Morphology & Count

Photomicrograph IV-16.Semen showing normal sperm count and motility without staining (x100)

134

Group I. Control

Normal Sperm Morphology & Count

Photomicrograph IV-17.Control semen showing normal sperm count and motility without staining (x400)

135

Group I. Control

Normal Sperm Morphology & Count

Photomicrograph IV-18.Controlsemen showing normal sperm count on Leishman’s stain on (x400)

136

GROUP I. CONTROL

Normal Sperms Count & Morphology

Photomicrograph IV-19.Semenshowing normal sperms count & morphology. (H&E on x400)

137

GROUP I. CONTROL

Normal Sperms Count & Morphology

Photomicrograph IV-20.Slide showing normal sperms count & maturation of sperms. (H & E x100)

138

GROUP I. CONTROL

Normal Sperms Count & Morphology

Photomicrograph IV-21.Slide showing normal sperms count & maturation of sperms. (H & E x200)

139

Group II. rhIFN- α-2b (3 mIU)

Decreased Sperm Count & Pre-mature Forms

Photomicrograph IV-22. Semen showing low sperm counts with pre mature forms on Leishman’s stain on (x100)

140

Group II. rhIFN- α-2b 3 mIU

Decreased Sperm Count & Pre-mature Forms

Photomicrograph IV- 23. Semen showing low sperm counts with premature forms without staining (x400)

141

Group II. rhIFN- α-2b 3 mIU

Low Sperm Count, Mixed Premature & mature Forms

Photomicrograph IV-24.Low sperm counts, mixed premature & mature forms of sperms.(H& E x100)

142

Group II. rhIFN- α-2b 3 mIU

Low Sperm Count, Short tail

Germinal epithelial cells

Photomicrograph IV-25. Low sperm counts with short tail with presence of primitive germ cell germinal epithelial cells. (H & E x400)

143

Group III. rhIFN- α-2b 5 mIU

Pre-mature Forms

Photomicrograph IV- 26.Premature forms of sperms without staining (x400)

144

Group III. rhIFN- α-2b 5 mIU

Abnormal Head & Tail of Sperm

Photomicrograph IV-27. Abnormal forms of sperms with abnormal head & tail. (H & E x400)

145

Group III. rhIFN- α-2b 5 mIU

Plasma Cells

Abnormal Sperms

Photomicrograph IV-28. Abnormal sperms with premature form, plasma cells. (H & E x200)

146

Group III. rhIFN- α-2b 5 mIU

Abnormal & Pre-mature sperms

Photomicrograph IV-29. Abnormal & premature forms of sperms with plasma cells. (H & E x400)

147

Group III. rhIFN- α-2b 5 mIU

Few Mature & immature forms

Photomicrograph IV-30. Few mature sperms with predominately immature forms of sperms. (H & E x400)

148

Group IV. rhIFN- α-2b 10 mIU

Primitive Forms of sperms

Photomicrograph IV- 31.Slide showing primitiveforms of sperms.

Leishman’s staining (x400)

149

Group IV. rhIFN- α-2b 10mIU

Pre-mature Sperms

Photomicrograph IV-32. Premature forms of sperms. (H & E x 100)

150

Group IV. rhIFN- α-2b 10mIU

Sperm with Short Tail

Photomicrograph IV-33. Sperms with short tails(H & E x200)

151

Group IV. rhIFN- α-2b 10mIU

Pre-mature Forms sperms & Plasma Cells

Photomicrograph IV-34. Premature forms of sperms and plasma cells (H & E x200)

152

Group IV. rhIFN- α-2b

10mIU

Plasma Cells

Pre-mature Forms of Sperms

Photomicrograph IV-35. Premature form of sperms and plasma cells (H & E x 400)

153

CHAPTER V DISCUSSION

Interferons (IFNs) are cytokines proteins having potential of anti viral, anti proliferative and immune regulatory functions. (93, 94) and are categorized as IFN types I, II and III according to antigenic and Physico-chemical properties and biological activity (95-97).

Type-I IFNs comprise of subtypes alpha (INF- α) and beta (INF-β). These are extensively used as therapeutic agents in the treatment of cancer and viral hepatitis B and C (98-100).

Side effects of recombinant human interferon (rhINF- α) vary from flu, nausea, anorexia and fever (101), to endocrine disorders such as thyroiditis (102) and the sexual dysfunction (103).

Presence of IFN-α in seminal plasma might have impact on sperm production since men with oligozoospermia show higher concentration of IFN-α in compared to normozoospermic males (104).

Transgenic male mice exposed to IFNs showed bizarre changes in spermatogenesis and eventually became sterile. Gonadal steroidogenesis is inhibited by IFN-γ in both in-vitro and in-vivo experiments, however the underlying mechanisms are not clearly elucidated (5-7).

In cultured cells, IFN-α is produced by peritubular myoid cells and Sertoli cells, as well as by germ cells. In contrast, it is evident that the IFN-γ is produced by early spermatids (8, 9).The IFN-α and IFN-γ receptors are expressed on the cell membrane of sperm cells of mammals during sperm formation in the seminiferous tubules. The expression of IFN-α and IFN-γ receptors show that the

IFNs may have implications in anti-sperm vaccine contraception and male

154 infertility. Gene mutations in controlled targeted studies have shown IFN inhibit development of germ cells in testes (3, 10).

Over expression of either the IFN α or IFN-ß gene disrupts the spermatogenesis and eventual destruction of spermatogonia in the testes of transgenic mice model (5, 6). Serum testosterone and free androgens are reduced when IFN-α was administered in healthy male (11, 12). Previous studies have reported that IFN–γ induces deleterious effects on testes histology with desquamation of germinal epithelium, and height of germinal cells, seminiferous tubule diameter and Sertoli cells are all reduced (3, 10).

The effects of IFN-α on testicular morphology and functions have never been clearly elucidated. Animal studies have shown conflicting results. In one study, the expression of IFN-α in transgenic mice revealed degeneration of spermatogonia with complete atrophy of seminiferous tubules (11). While other studies had reported that subcutaneous injections of IFN-α increased sperm production and serum testosterone levels in rodent models (13, 14).

The testosterone plays an important role in the spermatogenesis directly and indirectly through conversion into estrogen. The estrogen is essentially involved in sperm maturation in their terminal stages. The testosterone is converted into estrogens by the aromatization, the reaction being catalyzed by the action of p450-aromatase enzyme. The aromatase enzyme (AE) is expressed in the testis, liver and brain tissues. The aromatase enzyme gene (AEG) may be affected by drugs like morphine and recombinant human interferon-α-2b (rh-INF-

α-2b) which needs to be investigated also (15).

Most of the work in literature, concerning effects of interferons addresses the hormonal, and more generally the homeostatic effects of these drugs. The

155 phenotypic effects of these drugs at histological levels on testicular tissue have been seldom addressed. As currently Pakistan has much burden of viral hepatitis for which ideal curable drug being in use is the recombinant human interferon-α-

2b (rhINFα-2b), but its effects on testicular morphology, hormone production and aromatase gene remains unknown.

Cytokines and human reproduction are the new areas demanding research since cytokines are responsible for many aspects of infertility (105). A number of studies (105- 107) have evaluated the effects of IFN on biological systems; however, there are only a few studies as regards the use of rhINFα-2b on the male reproductive system (106), this compelled us to conduct the present study for addressing this issue.

The present study investigated the sperm parameters, serum sex hormones, testicular morphology in adult male rats exposed to rhINFα-2b for 30 days (107- 109).

In present study serum testosterone, serum FSH and LH were decreased in experimental rats compared to controls. 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 experimental groups II, III and IV respectively (p≤ 0.011) (table IV-1, IV-2 and graph IV-1).

Serum FSH reduced progressively in interferon treated animals at different doses compared to controls. 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) (table IV-3 and graph IV-2).

Serum LH also showed a reduction in interferon treated animals at different doses compared to controls. Serum LH, mean± SD, in control groups was 7.56±

156

0.70 compared to 6.8±0.63, 5.65±0.92 and 4.7±1.31 mIU/ml respectively (p≤

0.003) (table IV-4 and graph IV-3).

Testicular hormones are important in development of male reproductive organs, normal functioning of testes and accessory sex glands and in maintaining the spermatogenesis and fertility (107, 110, 111), hence a reduction in serum

Testosterone, serum FSH and serum LH might adversely affect these physiological processes (112). Our findings of low serum testosterone, serum

FSH and serum LH indicates interference of rhINFα-2b with male reproductive system and is consistent with above mentioned study.

A previous study by Andrade (113) reported that the INF-α is one of the possible endocrine disruptors. Andrade et al (113) reported that IFN-α induced effects on testosterone secretion in health and disease remain inconclusive because of different results, and this was reported to be due to the different experimental study protocols (95).

Previous studies (95, 114) demonstrated in human studies that the rh IFN-

α 2b injected subcutaneously resulted in a decrease in serum testosterone. The finding of reduced serum testosterone in response to rh IFN-α 2b is a consistent finding with our present study.

Yet another previous study in human beings reported that the rh IFN-α 2b reduced free androgen index in humans which was not associated with any change in gonadotropins (LH and FSH) (102).

The finding of serum FSH and LH is an inconsistent finding of Crossmit et al (102)as in present study a reduction was noted in both hormones. Serum FSH reduced progressively in interferon treated animals at different doses compared to controls. Serum FSH, mean± SD, in control groups was 7.43± 0.79 compared to

157

6.6±0.75, 6.02±0.82 and 5.2±1.18 mIU/ml respectively (p≤ 0.026) (table IV-3 and graph IV-2). Serum LH also showed a reduction in interferon treated animals at different doses compared to controls. 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 mIU/ml respectively (p≤ 0.003) (table IV-4 and graph IV-3). These differences might be most probably due to different study protocols, different animal models such as rats vs. mice, different hormonal assay procedure, and nonetheless research bias.

A previous study (115) conducted on patients with chronic hepatitis C who received doses of IFN-α2b and IFN-α2b combined with ribavirin 3 times a week for 12 months. This previous study reported marked reduction in serum testosterone which remained low even after cessation of therapy. The finding is consistent to the present study. They(115)reported no change in serum LH levels which is in contrary to present study as low LH was noted in present study.

Another previous study (116) reported a human study of chronic hepatitis C patients. They were treated with IFN-α, 3 times per week for 12 months. Serum testosterone levels were measured after every 3 months. The finding is in contradistinction to our present study and previous (106, 107). However, a mild reduction in serum FSH and LH were reported by previous study which is a consistent finding.

Another previous study(117)reported no marked change in serum LH and testosterone levels in patients on rhIFN-α therapy. Serum LH and testosterone levels were measured after every two months and rhIFN-α was injected thrice a week. The finding is contrary to our present and previous (106, 107) studies.

A previous research(107) reported study on adult Wistar rats from

Biotheriumv of the Morphology Department, Institute of Biosciences of Botucatu,

158

UNESP, Brazil. Rats were injected IFN-α at doses of 5 mIU and 10 mIU daily for

30 days. Serum testosterone, LH, FSH and testicular histology of rats were evaluated. Serum LH and FSH showed non-significant differences between controls and experimental rats. While serum testosterone showed a reduction as evident from their graphical presentation but was reported statistically non significant. The finding of serum LH and FSH is in contrast to present study while that of serum testosterone is consistent finding to present study. Rosa et al reported statistically non significant changes in testicular histology but minor changes were reported. Testicular morphometric and histomorphological changes of present study are in contrast to above study. Seminiferous tubules were thin in experimental rats compared to controls. Thickness and length of seminiferous tubules was reduced and diameter was increased in experimental groups.

Germ cell counts were reduced in experimental rats compared to controls.

The finding is also in contrast to Rosa et al. Present study noted germ cell maturation arrest and desquamation in experimental rats compared to controls.

Thick BM was found in more numbers of experimental rats compared to controls. 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 and Leydig cell counts were noted in the rhIFN treated experimental rats as shown in Table IV-17 and graph IV-16. This shows the INF impairs hormone production at peripheral and central level affecting the hypothalamo- pituitary- gonadal axis.

An experimental study (118)reported that injections of IFN-α daily for 3 months increased testosterone levels in male rats. The finding of Hibi et al is in contrast to present study and to previous study (107).

159

Another previous study by Mageed et al(106) reported raised serum testosterone levels in adult mice that were treated with intraperitoneal injections of

IFN-α-2b once a week for one three months. The finding of Mageed et al is in contrast to present study and to previous study(107).

The reason for such variations is clear from the protocol of IFN therapy by

Mageed et al, who injected IFN-α-2b just one a week. And experimental animals were mice contrary to Wistar rats of present study and previous study (107).

Finding of low serum LH of present study is in parallel to previous studies

(106,118), they reported a decrease in LH levels.

Finding of low serum FSH of present study is in parallel to previous studies of Mageed et al(106), who reported a decrease in FSH but contrary to Hibi et al

(118) reported no marked changes in FSH levels.

Normal functioning of testes is regulated by functional hypothalamic- pituitary-testicular axis, via a feedback mechanism which is in turn regulated by testosterone, LH, and FSH. LH and FSH are secreted by anterior pituitary under the influence of gonadotropin hormone-releasing hormone (GnRH). Leydig cells secrete testosterone under the action of LH, while Sertoli cells are acted upon by

FSH regulating the spermatogenesis (119). IFN-α administration leads to adverse male reproductive effects.

Sperm morphology of controls, rhIFN 3 MIU and 5 MIU showed abnormal morphology. 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 (Photomicrographs IV-15 to IV-28.). The findings are consistent to previous studies (107, 120- 123).

But some other studies had reported increased sperm counts and epididymal sperm counts. It was reported that the IFN-α does not alter sperm

160 production (118, 124). Findings are in contrast to present and previous studies

(106, 107).

Two previous studies evaluated the effects of IFN-γ on germinal epithelium in mice model. Both studies reported altered germinal epithelium and decreases spermatogenesis (125, 126). The findings of above studies are in parallel to present study as decreased germ cells were noted as shown in table IV-13.

Reduction of germ cell findings is also in parallel to previous studies, who reported altered spermatogonia and sterility in male mice treated with IFNs (127,

128).

IFNs inhibit gonadal steroidogenesis in both vivo and in vitro has been reported by previous studies (106, 107, 129, 130). The findings of decreased serum testosterone is a consistent finding supported by above cited studies.

Previous studies had reported unclear mechanism by which the INF-γ inhibits

Leydig's cells steroidogenesis.

But from the findings of decreased serum testosterone, serum FSH and

LH, the present study logically reports that the IFNs inhibit steroidogenesis at both central and peripheral level. IFNs disrupt the hypothalamo- pituitary- gonadal axis.

Mammalian sperm cells show expression of IFN-alpha and IFN-gamma receptors, which seem to develop during spermatogenesis. These findings may have implications in male infertility and anti-sperm contraceptive vaccine development (131).

In transgenic mice over expressing either the IFN or IFNß gene, the process of normal germ cell development (spermatogenesis) is disrupted with concomitant destruction of germ cells (128, 129).

161

The finding of germ cells is a consistent observation to present study because germ cells were reduced and their maturation was arrested in present experimental rat model study.

Effects of rh-INF-α-2b on gross anatomy of testicles have seldom been addressed, hence the present work was designed on testicular tissue and assessing effects on serum testosterone and gonadotropins levels.

In 2003 Chen et al (132) proposed that one possible mechanism of inhibition of Leydig's cell steroidogenesis by IFN, is that it down regulate the expression of glucose transport 8 (GLUT8), which results in decreased glucose uptake by Leydig's cells and inhibition of testosterone production. In present study serum testosterone was reduced by rh-INF-α-2b therapy.

A previous study (126) reported deleterious effects of INF on the histology of testes in mice. They (126) reported shortened height of germ cells, small tubular diameter and “desquamation: of the germinal epithelium. These findings are consistent to present study. However, they (126)reported significant decrease in the number of Sertoli cells which is in contrast to present study as increased

Sertoli cells were noted as shown in table IV-17 and graph IV-16.

Contrary report has been made by previous study (133) of increased testicular spermatogenesis and increase epididymis sperm concentration in the rat treated with INF. The finding is in contrast to present and previous studies (106,

107).

Corssmit et al (134) performed a crossover study in 6 healthy male. Serum concentrations of gonadotropins, testosterone, the free androgen index and sex

162 hormone-binding globulin (SHBG) rh-IFN-α- 2b. A sustained decrease in testosterone and free androgen index, and no change in concentrations of LH

(Luteinizing hormone), FSH (follicle stimulating hormone) and SHBG (sex hormone binding globulins) were noted. It was concluding that the INFs interfere at the level of the hypothalamic-pituitary- gonadal (testicular) axis at the testicular level.

Reduced serum testosterone of Crossmit et al (134) is consistent to present study. While Crossmit et al (134) has reported inconclusive results for the serum LH, FSH and SHBG.

On contrary, another study (135) reported seven male inflicted with chronic viral hepatitis. The rh-IFN-α- 2b was not proved of affecting sex hormones including the LH (luteinizing hormone), testosterone and free testosterone and the

SHBG (sex hormone-binding globulin).

The findings are in contrast to present study. The contrasting might be due to low sample size and low dose of rh-IFN-α- 2b used in male population. Findings are also in contrast to previous studies (106, 107, 134).

Fujisawa et al (104) investigated the role of rh-IFN-α in the seminal plasma on spermatogenesis in 101 male populations. No significant alterations were noted in the serum testosterone, LH, FSH, Prolactin and estradiol. The findings are in contrast to present and previous studies(106, 107, 134). It was postulated that the low seminal rh-IFN-α concentration might be responsible for no alterations in serum hormone levels and sperm production.

The present work confirmed that rh-IFN-α has inhibitory effects on testosterone, LH, FSH and deleterious effects on testicular anatomy. Leydig`s cells were increased in rh-IFN-α treated experimental rats as shown in table IV-

18. Our findings are in full agreement with Fujisawa et al(104)and Brown et

163 al(136). Both studies reported increased Leydig`s cells due to release of negative feedback of Hypothalamo pituitary testicular axis.We report the interactions between the immune system and reproductive systems. In addition, measuring the testicular tissue for interferon entry / residues will be worth empirical to confirm our findings.

164

CHPATER VI CONCLUSION

The present study concludes that the 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 abnormalities in interferon treated rats. Recombinant human IFN-

α2b administered at 3, 5 and 10 mIU caused marked effects on sperm quality and could also affect theof male rats.

165

CHPATER VII

RECOMMENDATIONS

Use of rh-INF-α-2b should be practiced with caution by clinicians. Sexual dysfunction should be strictly watched in patients receiving rh-INF-α-2b. Further studies are recommended to be coducted on animal models and also the human beings for making proper guidelines for rh-INF-α-2b use in clinical practice.

166

REFERENCES

1. El-Kady MAMH. Interferon-alpha signaling pathway in the sensory auditory neuroepithelial cells. (Thesis). University of Pittsburgh 2005. Accessed onlinewww.google.com.october15th2015.

2. Kalvakolanu DV, Borden EC. An overview of the interferon system: signal transduction and mechanism of action. Cancer Invest 1996; 14: 25–53.

3. Natwar RK, Mann A, Sharma RK, Aulitzky W, Frick J. Effect of human gamma interferon on mice testis: a quantitative analysis of the spermatogenic cells. Acta Eur Fertil 1995; 26: 45–49.

4. Bussiere JL, Hardy LM, Hoberman AM, Foss JA, Christian MS. Reproductive effects of chronic administration of murine interferon-gamma. Reprod Toxicol 1996; 10, pp: 379–791.

5. Hekman ACP, Trapman J, Mulder AH, van-Gaalen JL, Zwarthoff EC. Interferon expression in the testes of transgenic mice leads to sterility. J Biol Chem 1988;263: 12151–5.

6. Iwakura Y, Asano M, Nishimune Y, Kasade Y. Male sterility of transgenic mice carrying exogenous mouse interferon-ß gene under the control of the metallothionein enhancer-promoter. EMBO J 1988; 7:3757–3762.

7. Orava M, Cantell K, Vihko R. Treatment with preparations of human leukocyte interferon decreases serum testosterone concentrations in men. Int J Cancer. 1985; 38: 295–296.

8. Meikle AW, Cardoso DE, Sousa JC, Dacosta N, Bishop DK, Samlowski WE. Direct and indirect effects of murine interleukin-2, gamma interferon, and tumor necrosis factor on testosterone synthesis in mouse Leydig cells. J Androl 1992;13: 437–43.

9. Deujcq N, Dugast I, Ruffault A, Meide PH, Jegou B. Interferon-α and -γ expression in the rat testis. Endocrinology 1995;136:4925–4931

10. Mageed NA, Hassan E, Hegazy A, Wahab NMA, Ismail SA. Reproductive effects of human interferon alpha 2b administration on male albino mice testes: An Experimental study. Egypt J Hospt Med 2005; 19: 67-8.

11. Naz RK, Chauhan SC, Rose LP. Expression of alpha and gamma interferon receptors in the sperm cell. Mol Reprod Dev 2000; 56 (2): 189-97.

12. Corssmit EP, Endert E, Sauewein HP, Romijn JA. Acute effects of interferon-alpha administration on testosterone concentrations in healthy men. Eur J Endocrinol 2000; 143 (3): 371–74.

13. Hibi H, Yokoi K, Yamamto M. Effects of alpha-interferon on sperm production, concentration, and motility in the rat. PIMD 2004; 94: 77.

167

14. Longo I, Sanchez-Mateos P, Lazaro P, Longo N. Azoospermia in a patient receiving interferon alpha for a stage III melanoma. Acta Derm Venereol 2002; 82: 389-90.

15. Aloisi AM, Ceccarelli I, Fiorenzain P, Maddalena M, Rossi A, Tomei V, et al. Aromatase and 5-alpha reductase gene expression: modulation by pain and morphine treatment in male rats. Molecular Pain 2010; 6:69.

16. Sinnatamby CS. Last`s Anatomy: Regional and Applied. 11th edition. Churchill Livingstone, Elsevier, Edinburgh, Philadelphia 2009: 328-332.

17. Snell RS. Clinical Anatomy by Regions. 9th Edition. Lippincott Williams & Willkins, Wolters Kluwer Business, Philadelphia 2012: 275-277.

18. Tank PW. Grant`s Dissector. 14th Edition. Lippincott Williams & Willkins, Wolters Kluwer Health, Philadelphia 2009: 275-277.

19. Waugh A, Grant A. Ross & Wilson Anatomy and Physiology in Health and Illness. 10th edition. Lippincott Williams & Willkins, Wolters Kluwer Business, Philadelphia 2009: 441-450.

20. Barret KE, Barman SM, Botano S, Brooks H. Ganong`s Review of Medical Physiology, 24th ed. M`c Graw Hill Medical Publishing. New York. 2012: 428- 37.

21. Guyton AC, Hall JE. Textbook of Medical Physiology, 11th ed. Elsevier Saunders. Philadelphia, Pennsylvania. 2012: 996-9.

22. Lu W. Understanding Aromatase: A mechanistic basis for drug interactions and new inhibitor. Thesis: Doctor of Philosophy, Faculty of the University Graduate School, Department of Pharmacology and Toxicology, Indiana University 2012.

23. Weinbauer GF, Luetjens CM, Simoni M, Nieschlag E. Physiology of testicular function: Andrology. 5th Edition. Springer-Verlag Heidelberg. 2010: 11-59.

24. Luetjens CM, Didolkar A, Kliesch S, Paulus W, Jeibmann A, Bocker W, et al. Tissue expression of the nuclear progesterone receptor in male non-human primates and men. J Endocrinol 2006; 189: 529–539.

25. Modi DN, Shah C, Puri CP. Non-genomic membrane progesterone receptors on human spermatozoa. Soc Reprod Fertil 2007; 63: 515–529.

26. Simpson ER. Sources of estrogen and their importance. J Steroid Biochem Mol Biol 2003; 86 (3-5): 225-30.

27. Simpson ER, Davis SR. Minireview: aromatase and the regulation of estrogen biosynthesis--some new perspectives. Endocrinology 2001;142 (11): 4589-94.

28. Sebastian S, Bulun SE. A highly complex organization of the regulatory region of the human CYP19 (aromatase) gene revealed by the Human Genome Project. J Clin Endocrinol Metab 2001; 86 (10): 4600-2.

168

29. Bulun SE, Simpson ER. Aromatase expression in women's cancers. Adv Exp Med Biol 2008; 630: 112-32.

30. Santen R, Brodie JH, Simpson ER, Siiterri PS, Brodie A. History of aromatase: saga of an important biological mediator and therapeutic target. Endocr Rev2009; 30 (4): 343-75.

31. Wang H, Li R, Hu Y. The alternative noncoding exons 1 of aromatase (Cyp19) gene modulate gene expression in a posttranscriptional manner. Endocrinology 2009; 150 (7):3301-7.

32. Vaz AND. Drug metabolizing enzymes: cytochrome P450 and other enzymes in drug discovery and development. Fontis Media 2003.

33. Issacs A, Lindenmann J. Virus Interference. I. The interferon. Proc Royal Soc Lond (Biol) 1957; 147: 258-267.

34. Nagano Y, Kojima Y, Sawai Y. Immunity and interference in vaccinia; inhibition of skin infection by inactivated virus.C R Seances Soc Biol Fil 1954’148 (7-8): 750-752.

35. Knight E Jr. Interferon: purification and initial characterization from human diploid cells. Proc Natl Acad Sci USA 1976; 73(2): 520-23.

36. Berg K, Heron I. The complete purification of human leucocyte interferon. Scand J Immunol 1980; 11 (5), pp: 489-502.

37. Weck PK, Apperson S, May L, Stebbing N. Comparison of the antiviral activities of various cloned human interferon-alpha subtypes in mammalian cell cultures. J Gen Virol 1981; 57(Pt 1): 233-237.

38. Dworkin-Rastl E, Dworkin MB, Swetly P. Molecular cloning of human alpha and beta interferon genes from Namalwa cells. J Interferon Res 1982;2 (4): 575-85.

39. Gray PW, Goeddel DV. Cloning and expression of murine immune interferon cDNA. Proc Natl Acad Sci USA 1983; 80 (19): 5842-6.

40. Neumann-Haefelin D, Sundmacher R, Sauter B, Karges HE, Manthey KF. Effect of human leukocyte interferon on vaccinia-and herpes virus-infected cell cultures and monkey corneas. Infection and Immunity 1975;12:148-155

41. McGill JI, Collins P, Cantell K, Jones BR, Finter NB. Optimal schedules for use of interferon in the corneas of rabbits with herpes simplex keratitis. J Infect Disease 1976; 133 (Suppl): A13-17.

42. Trousdale MD, Robin JB, Strebbing N. Reevaluation of human alpha interferons against herpesvirus infection of the rabbit eye. Curr Eye Res 1987; 6: 269-272.

169

43. De-Koning EW, van-Bijsterveld OP, Cantell K. Combination therapy for dendritic keratitis with acyclovir and alpha-interferon. Arch Ophthalmol 1983; 101 (12): 1866-1868.

44. Sundmacher R, Cantell K, Mattes A. Combination therapy for dendritic keratitis. High-titer alpha-interferon and trifluridine. Arch Ophthalmol 1984;102: 554-5

45. Taylor JL, Punda-Polic V, O`Brian WJ. Combined anti-herpes virus activity of nucleoside analogs and interferon. Curr Eye Res. 1991; 10(Suppl):205-11

46. Ho M. Interferon as an agent against herpes simplex virus. J Invest Dermatol 1990;95:158-160

47. Jamesson P, Essex M. Inhibition of feline leukemia virus replication by human leukocyte interferon. Antiviral Res 1983; 3(2):115-20.

48. Fulton RW, Burge LJ. Susceptibility of feline herpesvirus 1 and a feline calicivirus to feline interferon and recombinant human leukocyte interferons. Antimicrob Agents Chemother 1985;28(5): 698-9.

49. Weiss RC. Synergistic antiviral activities of acyclovir and recombinant human leukocyte (alpha) interferon on feline herpesvirus replication. Am J Vet Res 1989; 50(10): 672-7.

50. Weiss RC, Oostrom-Ram T. Effect of recombinant human interferon-alpha in vitro and in vivo on mitogen-induced lymphocyte blastogenesis in cats. Vet Immunol Immunopathol 1990; 24(2): 147-57.

51. Eltern M. Efficacy of rHuIFN-α2b and rFeIFN-ω on Feline Herpesvirus-1 Replication in vitro. Thesis: Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA, USA 2005.

52. Haque SJ, Williams BR. Identification and characterization of an interferon (IFN). Molecular Cell Biology 1989; 9(11): 4605–4612.

53. Colonna M, Krug A, Cella M. Interferon-producing cells, on the front line in immune responses against pathogens. Current Opinion in Immunology 2002; 14:373.

54. Petska S, Langer JA, Zoon KC, Samuel CE. Interferons and their actions. Annual Review of Biochemistry 1987; 56: 727-777.

55. Platanias LC, Uddin S, Domanskii P, Colamonici OR. Interferon-β selectively induces the interaction of α and βL-subunits of the type I interferon receptor. The journal of biological chemistry 1996; 271(39):23630–23633

56. Kotenko SV, Gallagher G, Baurin VV, Lewis-Antes A, Shen M, Shah NK. IFN-λ mediates antiviral protection through a distinct class II cytokine receptor complex. Nature in Immunology 2003; 4: 69–77.

170

57. Sheppard P, Kindsvogel W, Xu W, Henderson K, Schlutsmeyer S, Whitmore TE, et al. IL-28, IL-29 and their class II cytokine receptor IL-28R. Nature and Immunology 2003; 4: 63-68.

58. Vilcek J. Novel interferons. Nature Immunology 2003; 4: 8-9.

59. Major ME, Dahari H, Mihalik K, Puig M, Rice CM, Neumann AU. Hepatitis C virus kinetics and host responses associated with disease and outcome of infection in chimpanzees. Hepatology 2004;39: 1709–1720.

60. Tan H, Derrick J, Hong J, Sanda C, Grosse WM, Edenberg HJ. Global transcriptional profiling demonstrates the combination of type I and type II interferon enhances antiviral and immune responses at clinically relevant doses. Journal of Interferon and Cytokine Research 2005;25(10):632-49

61. Thimme R, Bukh J, Spangenberg HC, Wieland S, Pemberton J, Steiger C, et al. Viral and immunological determinants of hepatitis c virus clearance, persistence, and disease. The proceedings of the National Academy of Sciences of USA 2002; 99:15661–15668.

62. Ihle JN, Witthuhn B, Tang B, Yi T, Quelle FW. Cytokine receptors and signal transduction. Baillieres Clinical Haematology 1994;7 (1): 17–48.

63. Leonard WJ. Role of Jak kinases and STATs in cytokine signal transduction. International Journal of Hematology 2001; 73 (3): 271-277.

64. Jacobs LD, Cookfair DL, Rudick RA, Herndon RM, Richert JR, Salazar AM, et al. Intramuscular interferon beta-1a for disease progression in relapsing multiple sclerosis. The Multiple Sclerosis Collaborative Research Group (MSCRG). Annals of Neurology 1996; 39 (3): 285-94.

65. Biron CA. Initial and innate responses to viral infections—pattern setting in immunity or disease Current Opinion in Microbiology 1999; 2:374-381.

66. York JD, Odom AR, Murphy R, Ives EB, Wente SR. A phospholipase C- dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export. Science 1999; 285: 96–100.

67. Cella M, Facchetti F, Lanzavecchia A, Colonna M. Plasmacytoid dendritic cells activated by influenza virus and CD40L drive a potent TH1 polarization. Nature Immunology 2000; 1:305.

68. Biron CA. Role of early cytokines, including and interferons (IFN-/), in innate and adaptive immune responses to viral infections. Seminars in Immunology 1998; 5: 383-390.

69. Zhang X, Sun S, Hwang I, Tough DF, Sprent J. Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15. Immunity 1998; 8: 591-599.

171

70. Durbin JE, Hackenmiller R, Simon MC, Levy DE. Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell 1996; 84: 443-450.

71. Davendra D, Eisenbarth GS. Interferon alpha—a potential link in the pathogenesis of viralinduced type 1 diabetes and autoimmunity. Clinical Immunology 2004; 111: 225–233. 72. Lande R, Giacomini E, Serafini B, Rosicarelli B, Sebastiani GD, Minisola G, et al. Characterization and recruitment of plasmacytoid dendritic cells in synovial fluid and tissue of patients with chronic inflammatory arthritis. Journal of Immunology 2004; 173: 2815–2824.

73. Nestle FO, Conrad C, Tun-Kyi A, Homey B, Gombert M, Boyman O, et al. Plasmacytoid pre-dendritic cells initiate psoriasis through interferon-alpha production. Journal of Experimental of Medicine 2005; 202(1):135-1343.

74. Ferbas JJ, Toso JF, Logar AJ, Navratil JS, Rinaldo CR. CD4+ blood dendritic cells are potent producers of IFN-alpha in response to in vitro HIV-1 infection. Journal of Immunology 1994; 152: 4649-4662.

75. Gottenberg JE, Cagnard N, Lucchesi C, Letourneur F, Mistou S, Lazure T, et al. Activation of IFN pathways and plasmacytoid dendritic cell recruitment in target organs of primary Sjögren's syndrome. Proceedings of the National Academy of Science of U S A 2006; 28(13): 5242.

76. Fulda S, Debatin KM. IFN- sensitizes for apoptosis by upregulating caspase-8 expression through the Stat1 pathway. Oncogene 2002; 21: 2295-2308.

77. Inaba H, Glibetic M, Buck S, Ravindranath Y, Kaplan J. Interferon- sensitizes osteosarcoma cells to fas-induced apoptosis by up-regulating Fas receptors and caspase-8. Pediatric Blood & Cancer 2004;43 (7):729-736.

78. Thyrell L, Erickson S, Zhivotovsky B. Mechanisms of IFN-alpha induced apoptosis in malignant cells. Oncogene 2002;21:1251–1262.

79. Dinarello CA. Biologic basis for interleukin-1 in disease. Blood 1996;87: 2095- 2147.

80. Browning JL, Ribolini A. Interferon blocks interleukin 1-induced prostaglandin release. Journal of Immunology 1987; 138:2857-2863.

81. Ren R, Jin H, Piao X, Piao F. Ribavirin and IFN-α combination therapy induces CD4+ T-cell proliferation and Th1 cytokine secretion in patients with chronic hepatitis B. World Journal of Gastroenterology 2007;13(41):5440-5445.

82. Heathcote J, Main J. Treatment of hepatitis C. Journal of Viral Hepatitis 2005; 12: 223-235.

83. Lisker-Melman M, Di Bisciglie AM, Usala SJ. Development of thyroid disease during therapy of chronic viral hepatitis with alpha interferon. Gastroenterology 1992; 102:2155-2160.

172

84. Marcellin P, Pouteau M, and Benhamou JP. Hepatitis C virus infection, alpha interferon therapy and thyroid dysfunction. Journal of Hepatology 1995; 22:364-369.

85. Benelhadj S, MarcellinP, Castelnau C. Incidence of dysthyroidism during interferon therapy in chronic hepatitis C. Hormonal Research 1997;48:209-14.

86. Becker JC, Winkler B, Klingert S. Antiphospholipid syndrome associated with immunotherapy for patients with melanoma. Cancer 1994;73:1621-4.

87. Poynard T, Bedossa P, Chevallier M, et al. A comparison of three interferon alfa-2b regimens for the long term treatment of chronic non-A, non-B hepatitis. New England Journal of Medicine 1995; 332: 1457-62.

88. Vial T, Descotes J. Clinical toxicity of the interferons. Drug Safety 1994; 10:115-150.

89. Stuart-Harris RC, Lauchlan R, Day R. The clinical application of the interferons: a review. NSW Therapeutic Assessment Group. Med J Aust 1992; 156 (12): 869-72.

90. Eltern M. Efficacy of rHuIFN-α2b and rFeIFN-ω on Feline Herpesvirus-1 Replication in vitro. Thesis: Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA, USA 2005.

91. Evans G, Maxwell WMC. Handling and examination of semen. In: Maxwell WMC (editor) Salmon`s artificial insemination of sheep and goat. Butterworth’s, Sydney. 1987: 83-106.

92. Gonzalez SE. Effect of methyl-tert-butyl ether (MTBE) on aromatase gene expression in female Sprague-Dawley rats. Thesis. Masters of Science. Department of Toxicology, Faculty of San Diego University, San Diego 2013.

93. Dejucq, N., Lienard, M. O., Guillaume, E.,Dorval, I., and Jégou, B. 1998. Expressionof interferons-alpha and -gamma in testicularinterstitial tissue and spermatogonia of therat. Endocrinology 139: 3081–87.

94. Brojeni, P. Y., Matok, I., Garcia Bournissen, F.,and Koren, G. 2012. A systematic review ofthe fetal safety of interferon alpha. Reprod.Toxicol. 33: 265–68.

95. Corssmit, E. P., de Metz, J., Sauerwein, H. P.,and Romijn, J. A. 2000a. Biologic responsesto IFN-alpha administration in humans. J.Interferon Cytokine Res. 20: 1039–47.

96. Randall, R. E., and Goodbourn, S. 2008.Interferons and viruses: An interplaybetween induction, signalling, antiviralresponses and virus countermeasures. J. Gen.Virol. 89: 1–47.

173

97. Gonzáles-Navajas, J. M., Lee, J., David, M., andRaz, E. 2012. Immunomodulatory functionsof type I interferons. Nat. Rev. Immunol. 12:125– 35.

98. Lechner, J., Malloth, N., Seppi, T., Beer, B.,Jennings, P., and Pfaller, W. 2008. IFN-alphainduces barrier destabilization and apoptosisin renal proximal tubular epithelium. Am. J.Physiol. Cell. Physiol. 294: 153–60.

99. Li, Z. L, Strunk, J. J., Lamken, P., Piehler, J.,and Walz, T. 2008. The EM structure of atype I interferon–receptor complex revealsa novel mechanism for cytokine signaling. J.Mol. Biol. 377: 715–24.

100. George, P. M., Badiger, R., Alazawi, W., Foster,G. R., and Mitchell, J. A. 2012. Pharmacologyand therapeutic potential of interferons.Pharmacol. Ther. 135: 44–53.

101. Lechner, J., Krall, M., Netzer, A., Radmayr, C.,Ryan, M. P., and Pfaller, W. 1999. Effects ofinterferon alpha-2b on barrier function andjunctional complexes of renal proximal tubularLLC-PK1 cells. Kidney Int. 55: 2178– 91.

102. Corssmit, E. P., Endert, E., Sauerwein, H. P.,and Romijn, J. A. 2000b. Acute effectsof interferon-alpha administration on testosteroneconcentrations in healthy men. Eur. J.Endocrinol. 143: 371–74.

103. Kraus, M. R., Schafer, A., Bentink, T.,Scheurlen, M., Weissbrich, B., Al- Taie, O.,and Seufert, J. 2005. Sexual dysfunctionin males with chronic hepatitis C andantiviral therapy: Interferon-induced functionalandrogen deficiency or depression? J.Endocrinol. 185: 345–52.

104. Fujisawa, M., Fujioka, H., Tatsumi, N., Inaba, Y.,Okada, H., Arakawa, S., and Kamidono, S.Levels of interferon alpha and gammain seminal plasma of normozoospermic,oligozoospermic, and azoospermic men.Arch. Androl 1998; 40: 211–14.

105. Cohen, D. R., Basu, S., Randall, J. M., Aballa,T. C., Lynne, C. M., and Brackett, N. L.2004. Sperm motility in men with spinal cordinjuries is enhanced by inactivating cytokinesin the seminal plasma. J. Androl. 25: 922–25.

106. Mageed N A, Hassan E, Hegazy A,Wahab NMA, Ismail SA.Reproductive effects of human interferonalpha-2b administration on male albino micetestes. An experimental study. Egypt. J. Hosp.Med 2005 19: 67–78.

107. Josiane de Lima Rosa, Marilia Martins Cavariani, Cibele dos SantosBorges, Gabriel Adan Araújo Leite, Janete Aparecida Anselmo- Franci & Wilma De GravaKempinas (2015) Lack of Reproductive Toxicity in Adult Male Rats Exposed to Interferon-Alpha, Journal of Toxicology and Environmental Health, Part A, 78:20, 1288-1298.

108. Heinlein, C. A., and Chang, C. 2002. Androgenreceptor (AR) coregulators: An overview.Endocr. Rev. 23: 175–200.

174

109. Smith, L. B., and Walker, W. H. 2014. Theregulation of spermatogenesis by androgens.Semin. Cell. Dev. Biol. 30: 2–13.

110. Luccio-Camelo, D. C., and Prins, G. S. 2011.Disruptor of androgen receptor signaling inmales by environmental chemicals. J. Steroid.Biochem. Mol. Biol. 127: 74–82.

111. Andrade, L. J. O. 2012. Interferron-alpha: Anendocrine disruptor? Arq. Bras. Endocrinol.Metab. 56: 72–73.

112. Orava, M., Cantell, K., and Vihko, R. 1986.Treatment with preparations of humanleukocyte interferon decreases serum testosteroneconcentrations in men. Int. J. Cancer38: 295–96.

113. Kraus, M. R., Schafer, A., Bentink, T.,Scheurlen, M., Weissbrich, B., Al- Taie, O.,and Seufert, J. 2005. Sexual dysfunctionin males with chronic hepatitis C andantiviral therapy: Interferon-induced functionalandrogen deficiency or depression? J.Endocrinol. 185: 345–52.

114. Piazza, M., Tosone, G., Borgia, G., Orlando, R.,Fenzi, G., Vitalee, M., Bloise, A., Mariano, A.,and Macchia, V. 1997. Long-term interferonalphatherapy does not affect sex hormonesin males with chronic hepatitis C. J. InterferonCytokine Res. 17: 525–29.

115. Barreca, T., Picciotto, A., Fransceschini, R.,Varagona, G., Garibaldi, A., Valle, F., Cataldi,A., D’Agostino, S., and Rolandi, E. 1993. Sexhormones and sex hormone-binding globulinin males with chronic viral hepatitis duringrecombinant interferon-alpha 2b therapy. J.Interferon Cytokine Res. 13: 209–11.

116. Hibi, H., Yokoi, K., and Yamamoto, M. 1997.Effects of alpha-interferon on sperm production,concentration, and motility in the rat.Int. J. Urol. 4: 603–7.

117. Amory, J. K., and Bremner, W. J. 2003.Regulation of testicular function in men:Implications for male hormonal contraceptivedevelopment. J. Steroid. Biochem. Mol.Biol. 85: 357–61.

118. Jensen, T. K., Bonde, J. P., and Joffe, M. The influence of occupational exposure onmale reproductive function. Occup. Med.2006; 56: 544–53.

119. Amory, J. K., and Bremner, W. J. 2003.Regulation of testicular function in men:Implications for male hormonal contraceptive development. J. Steroid. Biochem. Mol.Biol 2003; 85: 357–61.

120. Jensen, T. K., Bonde, J. P., and Joffe, M. The influence of occupational exposure onmale reproductive function. Occup. Med 2009; 56: 544–53.

121. Amann R P. Considerations in evaluatinghuman spermatogenesis on the basisof total sperm per ejaculate. J. Androl 2009; 30:626–41.

175

122. Perobelli, J. E., Martinez, M. F., Silva Franchi,C. A., Fernandez, C. D., Camargo, J. L., andKempinas, W. G. 2010. Decreased spermmotility in rats orally exposed to single ormixed pesticides. J. Toxicol. Environ. HealthA 73: 991–1002.

123. Fernandes, G. S., Favareto, A. P., Fernadez,C. D., Bellentani, F. F., Arena A. C., Grassi,T. F., Kempinas, W. G., and Barbisan, L. F.2012. Effects of diuron on male rat reproductiveorgans: A developmental and postnatalstudy. J. Toxicol. Environ. Health A 75:1059–69.

124. Oatley, J. M., and Brinster, R. L. 2012. Thegermline stem cell niche unit in mammaliantestes. Physiol. Rev. 92: 577–95.

125. Natwar RK, Mann A, Sharma RK, Aulitzky W, Frick J. Effect of human gamma interferon on mice testis: a quantitative analysis of the spermatogenic cells. Acta Eur Fertil 1995; 26:45–49

126. Bussiere JL, Hardy LM, Hoberman AM, Foss JA, Christian MS.Reproductive effects of chronic administration of murine interferon- gamma. Reprod Toxicol 1996; 10:379–391.

127. Hekman ACP, Trapman J, Mulder AH, van Gaalen JL, Zwarthoff EC.Interferon expression in the testes of transgenic mice leads to sterility. J Biol Chem 1988; 263:12151–12155.

128. Iwakura Y, Asano M, Nishimune Y, Kawade Y. Male sterility of transgenic mice carrying exogenous mouse interferon-ß gene under the control of the metallothionein enhancer-promoter. EMBO J 1988; 7:3757–3762.

129. Orava M, Cantell K, Vihko R. Treatment with preparations of human leukocyte interferon decreases serum testosterone concentrations in men. Int J Cancer 1985; 38:295–296.

130. Meikle AW, Cardoso de Sousa JC, Dacosta N, Bishop DK, Samlowski WE. Direct and indirect effects of murine interleukin-2, gamma interferon, and tumor necrosis factor on testosterone synthesis in mouse Leydig cells. J Androl 1992; 13:437–443.

131. Naz RK, Chauhan SC, Rose LP. Expression of alpha and gamma interferon receptors in the sperm cell. Mol Reprod Dev 2000; 56(2): 189- 197.

132. Chen T, Nagpal ML, Lin T. Expression and regulation of glucose transport 8 in rat leydig cells. J Endocrinol 2003; 179(1): 63-72.

133. Hibi H, Yokoi K, Yamamoto M. Effects of alpha-interferon on sperm production, concentration, and motility in the rat. PIMD 2004; 9477192

134. Corssmit EP, Endert E, Sauerwein HP, Romijn JA. Acute effects of interferon-alpha administration on testosterone concentrations in healthy men. Eur J Endocrinol 2000; 143(3): 371 – 374.

176

135. Barreca T, Picciotto A, Franceschini R, Varagona G, Garibaldi A, Valle F, Cataldi A, et al.Sex hormones and sex hormone-binding globulin in males with chronic viral hepatitis during recombinant interferon-alpha 2b therapy. J Interferon Res 1993; 13(3): 209-211.

136. Brown TT, Dobs A. Endocrine effects of marijuana. J. Clin. Pharmacol 2002; 42(11): 90-96.

Appendix

Proforma

Effects of Recombinant Human Interferon-α-2b on testicular morphology and testosterone production in albino rat model

Department of Anatomy, Isra University

1. Gross anatomy of Testes

2. Testicular morphometry

3. Testicular histomorphometry

4. Sperm Morphology

5. Testosterone level

6. Luteinizing hormone level

7. Follicle stimulating hormone level

8. Aromatase Gene study

APPENDIX- I

HAEMATOXYLIN AND EOSIN STAINED SECTIONS

In Hematoxylin and eosin (H & E) stained sections, general morphology of kidneys was observed. The tissues of testes were observed up to 40´ objective and 10´ ocular. Tissue sections were observed under 10´, 20´, 40´, 100´ objectives.

ROUTINE TISSUE PROCESSING

The tissues were processed as follows:

1. Fixation

The tissue was fixed in 10% formalin for H & E stains.

Composition of 10% Formalin

40% formaldehyde: 100 ml

Distilled water: 900 ml

2. Dehydration

Tissue was passed through a series in ascending strengths of ethyl alcohol, starting from 70%, 80%, 95% and two changes in absolute alcohol, 10 minutes for each step.

3. Clearing

Tissue was passed rapidly through xylene until tissue was cleared.

4. Infiltration

Tissue was infiltrated with molten paraplast at 58oC. Two changes were given, for 30 minutes each.

5. Casting (blocking or embedding)

The tissue was enclosed in a solid mass of paraplast. This was done with two L-shaped metal pieces. The cold L-shaped metal pieces were placed on a glass to produce squares of desired sizes. The enclosures were filled with melted paraplast. The tissue was placed in the squares in vertical position with the help of warm forceps. The blocks were labeled, allowed to cool, and the metal pieces were removed. The blocks were trimmed free of excess paraplast leaving some free margin around the embedded object.

6. Sectioning

Four micron thick vertical sections were cut on rotary microtome.

7. Fixation

The slides were placed on hot plate at 370C for 24 hours for proper fixation.

8. Staining

The slides were stained with H & E.

9. Mounting

The sections were mounted on cleaned gelatinized slide labeled with diamond pencil.

The stained slides after proper labeling were stored in special wooden boxes.

HAEMATOXIYLIN AND EOSIN STAIN

(BANCROFT AND STEVENS, 1982)

Preparation of stains

Harris’s Hematoxylin (Harris, 1900)

I. Hematoxylin :: 2.50gm II. Absolute Alcohol :: 25.00 ml III. Potassium alum :: 50.00 gm IV. Distilled Water :: 500.00 ml V. Mercuric Oxide :: 1.23 gm VI. Glacial Acetic Acid :: 2.0..ml

The Hematoxylin was dissolved in absolute alcohol. The alum was dissolved in warm distilled water and then added to the alcohol Hematoxylin. The mixture was rapidly brought to the boil and then mercuric oxide was added. The stain was rapidly cooled by plunging the flask into cold water. The glacial acetic acid was then added and the stain was ready for immediate use.

0.5% Eosin ‘Y’

I. Eosin ‘Y’ :: 0.5 gm II. Distilled water :: 5.0 ml III. Absolute ethyl alcohol :: 95 ml

The eosin ‘Y’ was dissolved in 5 ml distilled water and then added to 95 ml absolute alcohol.

ROUTINE HAEMATOXIYLIN & EOSIN STAINING PROCEDURE

(DELAFIELD’S (OR HARRIS) HAEMATOXYLIN)

S # Step Solution Time Function

Remove Xylene 2 min paraffin

1 Deparaffinization Xylene 2 min

Replace xylene Absolute alcohol 2min with alcohol

95% alcohol 2min

Replace 2 Hydration 80% alcohol 2 min alcohol with water

70% alcohol 2 min

3 Washing Water 2 min

Check after 1 Stain nuclei Delafield’s 4 Staining min for stain and other hematoxylin intensity basophilic parts

Several 5 Blueing Tap water changes (5-10 Blue nuclei min)

70% alcohol 2 min

Replace water 6 Dehydration 80% alcohol 2 min with alcohol

95% alcohol 2 min

About one Stain stain cytoplasm & 0.5% eosin in cytoplasm and 7 Counter stain other 95% alcohol other acidophilic acidophilic structures structures

Dehydrate and 95% alcohol 1-2 min also remove excess eosin 8 Dehydration Absolute alcohol 1-2 min

Absolute alcohol 1-2 min

9 Clearing Xylene 2 min

Xylene 2 min Replace alcohol with Mount in xylene and Canada Balsam 10 Mounting clear tissue or synthetic resin 9DPX)

Result:

Nuclei- Blue appearance

Cytoplasm- Pink coloration

APPENDIX- II

PREPARATION OF LEISHMAN STAIN

Requirements

• Leishman’s stain powder of high (at least 80%) Purity, 0.2g

• Methanol (acetone free), 100 ml

• Conical flask

• Funnel and filter paper

• Mortar and pestle

Preparation

• Weight 0.2g of powder stain and transfer it to a mortar.

• Grind with about 25 ml of methanol and allow it to settle. Transfer supernatant

through filter paper to the flask.

• Add another 25 ml of methanol to mortar containing residual stain. Repeat

grinding, allow to settle and transfer the supernatant to the flask.

• Repeat procedure until whole methanol has been used and most of the stain has

been dissolved.

• Place the flask in a water bath at 50˚C for 15 min.

• Filter into a clean brown borosilicate glass bottle for ripening.

• Leave to mature for at least 2-3days in the dark at room temperature.

A good practice is to make 2-3 bottles at a time initially. When one bottle is

finished, it should be replaced with freshly prepared stain and left to mature. In

the mean time other bottle of stain is used. Required volume of stain for daily use

should be filtered into a smaller dropping bottle every morning.

PREPARATION OF BUFFER

(SORENSEN’S 66 mmol/L)

Preparation

1. Solution A: Dissolve carefully weighed postassium dihydrogen phosphate in one

litre of distilled water in a conical flask, transfer to a clean glass bottle and store

in refrigerator.

2. Solution B: Dissolve and store disodium hydrogen phosphate in one litre of

distilled water.

3. To prepare buffer of pH 6.8, mix 50.8 ml of solution A with 49.2 ml of solution B

(page 420).

STAINING OF SEMEN FILMS WITH LEISHMAN STAIN

Requirement

• Prepared leishman stain

• Buffered water. Dilute 50 ml of Sorensen’s buffer of ph 6.8 to one liter with

distilled water.

• Staining rack

Procedure

• Prepare the semen film and air dry.

• Keep it on a staining rack and cover completely with stain.

• Leave to stain for 2 min.

• Pour buffered water on to the slide about twice the amount of stain. Mix by

blowing gently through a pipette. Leave for 5-7min.

• Pour of stain mixture. Wash in buffer, cleaning the underside of slide with a

tissue paper.

• Place vertically to drain and dry.

HAEMATOXYLIN AND EOSIN STAINING

It is commonly used for routine histopathology and in diagnostic cytology. Its particular value lies in its ability of imparting proper differentiation to distinguish between different types of connective tissue fibers and matrices, by staining them different shades of red and pink.

Principle:

First the tissue is rehydrated to facilitate the entry of dyes. The tissue section are then sequentially exposed to a basic dye e.g., eosin Harris’s Hematoxylin and an acid dye e.g., eosin. This stains both basic and acid components of the tissue.

Reagents

Harris’s Hematoxylin:

Hematoxylin crystals 5.0 g

Alcohol 95% 50ml

Ammonium or potassium Alum 100 g

Mercuric oxide 2.5 g

Distilled water 1 litre

Glacial acetic acid 40 ml

Dissolve separately by heating, haematoxylin in alcohol and alum in water, mix and rapidly boil. Remove from flame and add mercuric oxide. Reheat for 1 min or until becomes dark purple.

Remove from flame and cool in a basin of cold water. Stain is ready to use. Add 2-4 ml of Glacial acetic acid per 100 ml of solution if desired.

Acid alcohol: Mix one litre 70% alcohol with 10 ml of concentrated hydrochloric acid.

Ammonia water: Mix 2-3 ml of strong ammonia with one litre of tap water.

Alcoholic eosin solution:

Eosin (water soluble) 2g

Distilled water 160 ml

Alcohol 95% 640 ml

Other reagents: Xylol, absolute alcohol, rectified spirit and methylated spirit are also needed.

Staining procedure for Semen smear

1. Prepare the semen film and air dry.

2. Then transfer to absolute alchohol for 3 min.

3. Transfer to rectified spirit (80% alcohol) for 2 min.

4. Place in methylated spirit for 2 min.

5. Wash the slide in running water for 1 min and put it in Harris haematoxylin for 3-5

min.

6. Wash in running water for 30 seconds and wash the excess dye in 1% acid

alcohol by continuous agitation for 15 seconds.

7. Wash in running water for 30 seconds.

8. Give 2-3 dips in ammonia water solution until tissues attain a blue colour.

9. Wash in running water for 2-3 dips.

10. Counter stain with eosin for 2-3 min.

11. Wash in running tap water for 30 seconds.

12. Dehydrate by keeping in increasing concentrations of alcohol (2-3 dips in 70%,

95% and absolute alcohol).

13. Clear it in xylol and mount with Canada balsam.