DETERMINATION OF PRION PROTEIN (PrP) AND CHANGES IN FERTILITY HORMONES WITH SLEEP DEPRIVATION IN ALBINO RATS

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DETERMINATION OF PRION PROTEIN (PrP) AND CHANGES IN FERTILITY HORMONES WITH SLEEP DEPRIVATION IN ALBINO RATS

ABSTRACT

Wistar albino rats, numbering thirty five (35), were nurtured in the animal house of University of Nigeria Enugu Campus and used for this work. This work is designed to determine the presence of prion (PrP) in Wistar albino rats and the possible changes that sleep deprivation can cause in PrP and fertility hormones. Twenty four (24) of the animals (15 females 9 males) were successfully sleep-deprived for 14 days while 11 were not deprived of sleep. The non-sleep deprived rats were used as a control group in addition to PrPc commercial control, for the prion protein determination. The body weights of the rats were obtained before and after sleep deprivation. Serum samples were collected before and after sleep deprivation for the fertility hormone assay while brain tissues were extracted from each sleep deprived and non-sleep deprived rat after 14 days for prion protein determination and histological studies. Single platform sleep deprivation technique was used for sleep deprivation, ocular venipuncture for blood collection, euthanization for sacrificing the rats and enzyme linked immunosorbent assay method for both hormone assay and prion protein determination respectively. Part of the brain tissues were prepared histologically (sectioning and staining) using congo-red staining technique for possible sleep deprivation induced morphological changes. The presence of PrP as determined, was confirmed by comparison of the values of the two control groups and test samples while a significant increase (p < 0.05) in PrP concentration after sleep deprivation was observed when compared with non sleep deprived group of albino rats. Sex hormones such as testosterone and oestradiol, decreased significantly (p < 0.05). The concentrations of prolactin and thyroid stimulating hormone and the body weight of the rats also showed a significant decrease (p < 0.05) after sleep deprivation compared with the normal control rats. The concentrations of follicle stimulating hormone and luteinizing hormone had no significant (p > 0.05) changes after sleep deprivation when compared with control group of albino rats. There was decrease in oestradiol, testosterone, prolactin, thyroid stimulating hormones and body weight of rats while FSH, LH and brain tissues showed no significant changes. There were also no observable changes in the brain tissue morphology after sleep deprivation. In conclusion, there was PrPC induction following sleep deprivation in albino rats. It is therefore recommended that sleep deprivation should be put into consideration in infertility cases and more work should be done on Prion proteins for neuropathological cases.

TABLE OF CONTENTS

Title Page

Certification

Dedication

Acknowledgments

Abstract

Table of Contents

List of Figures

List of Plates

List of Abbreviations

CHAPTER ONE: INTRODUCTION

1.1 Sleep

1.1.1 Biology of Sleep

1.1.2 Regulation of Sleep

1.1.3 Functions of Sleep

1.2 Sleep Deprivation

1.2.1 Sleep Disorders

1.2.2 Sleep Deprivation and Associated Problems

1.2.3 Sleep Deprivation and Protein Metabolism

1.2.4 Sleep Deprivation and Prion Protein

1.3 Prion Protein (PrP)

1.3.1 Functions of Prion Protein

1.3.2 Prion Protein and Cell Membrane Viability

1.3.3 Prion Proteins and Sleep

1.3.4 Anti-Apoptotic Function

1.3.5 Protein and immune System

1.3.6 Prion Protein and Muscular Tone

1.3.7 Abnormal Prion Protein

1.3.8 The Pathogenicity of Prion

1.3.9 The Diseases of Prion (PrPres)

1.4 Endocrine System

1.5 Hormones

1.5.1 Follicle Stimulating Hormone

1.5.2 Luteinizing Hormones

1.5.3 Prolactin (PRL)

1.5.4 Thyroid Stimulating Hormone

1.5.5 Testosterone

1.5.6 Oestradiol

1.6 Body Weight

1.7 Analysis of Methodology for Sleep Deprivation, Prion Protein and Hormones Assay

1.7.1 Gentle Handling

1.7.2 Single Platform

1.7.3 Multiple Platforms

1.7.4 Modified Multiple Platforms

1.7.5 Pendulum

1.7.6 Automated sleep deprivation

1.7.7 Prion Protein Detection Methods

1.7.7.1 Western blot

1.7.7.2 Immunohistochemistry

1.7.7.3 ELISA

1.7.7.4 Staining of Amyloid Proteins

1.8 Nature of Research Subject

1.9 Consent

1.10 Aim and Objectives of the Study

1.10.1 Aim of the Study

1.10.2 Specific Objectives of the Study

CHAPTER TWO: MATERIAL AND METHODS

2.1 Materials

2.1.1 Animals

2.1.2 Chemicals and Reagents

2.1.3 Equipment

2.2 Methods

2.2.1 Sleep Deprivation

2.2.2 Blood Collection

2.2.3 Rat Sacrifice

2.2.4 EIA

2.2.4.1 EIA for Prion Protein Using Spi-Bio Kit

2.2.4.2 EIA for Follicle Stimulating Hormone (FSH)

2.2.4.3 EIA for Luteinizing Hormone (LH)

2.2.4.4 EIA for Prolactin

2.2.4.5 EIA for Thyroid Stimulating Hormone (TSH)

2.2.4.6 EIA for Testosterone

2.2.4.7 EIA for Oestradiol

2.2.5 Histological Procedure for Demonstration of Brain Tissue Morphology/Amyloid Protein

2.2.5.1 Alkaline Congo-red Method

2.3 Statistical Analysis

CHAPTER THREE: RESULTS

3.1 Prion Protein (PrP)

3.2 Follicle Stimulating Hormone Concentration of Control and Sleep Deprived Rats

3.3 Luteinizing Hormone (LH) Concentration of Control and Sleep Deprived Rats

3.4 Oestradiol (E2) Concentration of Control and Sleep Deprived Rats

3.5 Testosterone Concentration of Control and Sleep Deprived Rats

3.6 Prolactin Concentration of Control and Sleep Deprived Rats

3.7 Thyriod Stimulatine Hormone (TSH) Concentration of Control and Sleep Deprived Rats

3.8 Body Weight of Control and Sleep Deprived Rats

3.9 Brain Tissue Morphology of Control and Sleep Deprived Rats

CHAPTER FOUR: DISCUSSION

4.1 Discussion

4.2 Conclusion

4.3 Suggestions for Further Studies

REFERENCES

APPENDICES

LIST OF ABBREVIATIONS

AchE Acetylcholinesterase Enzyme

AIDS Acquired Immune Deficiency Syndrome

AMP Adenosine Monophosphate

BDI Benzodizpine

BIP Immunoglobulin Binding Protein.

BSE Bovine Spongyform Encephalopathy

CAH Congenital Adrenal Hyperplasia

cAMP Cyclic Adenosine Monophosphate

CCK Cholecystokinin

CCIP Corticotropin Intermediate Lobe Peptide.

CJD Creutzfeldt Jacobs Disease

CNS Central Nervous System

CSF Cerebrospinal Fluid.

CWD Chronic Wasting Disease

DHT Dihydrotestosterone

DNA Deoxyribonucleic Acid

DSIP Delta Sleep Inducing Peptide

E2 Estradiol

EEG Electroencephagram

EIA Enzyme Immuno Assay

ELISA Enzyme Linked Immunosorbent Assay

ER Endoplasmic Recticulum

FSH Follicle Stimulating Hormone.

fCJD Familial Creutzfeldt Disease

GC Glucocorticoid

GH Growth Hormone

GHRH Growth Hormone Releasing Hormone.

GPI Glycosyl phosphatidyl Inositol.

GRF Growth Releasing Factor

HCG Human Corionic Gondotrophin

HIV Human Immune Deficiency Syndrome

HP.A Hypothalamic Pituitary Axis

IFN Interferone

IGF Insulin Like Growth Factor

LH Luteinizing Hormone

MP Muramy Peptide

mRNA Messenger RNA

MSH Melanocyte Stimulating Hormone

NADPH Nicotinamide Adeninedinuclutide

NREM Non Rapid Eye Movement

OT Oxytocin

PGD2 Prostagladin D2

PL/PRL Prolactin.

PK Proteinase-K

PIH Prolactin Inhibiting Hormone.

PRNP/PrnP Human PrP gene

PrP Prion Protein.

PrPc Cellular Protein/Normal PrP

PrPsen Proteinase-K Sensitive PrP/Normal PrP

PrPsc Screpie PrP/Abnormal PrP

PrPres Proteinase-K Resistant PrP/Abnormal PrP

TSE Transmissible Spongyform Encephalopathy

PS Paradoxical Sleep

PSD Paradoxical Sleep Deprivation

PTH Parathyroid Hormone

PVN Paraventicular Nucleus

RAS Reticular Activity System

REM Rapid Eye Movement.

SDS Sodium Deodescyl Sulphate

SPS Sleep Promoting Substances

SWS Slow Wave Sleep

T3 Triiodothyronine

T4 Total Thyroxine

TMB Tetramethylbenzidine

TME Transmissible Mink Encephalopathy.

TNF Tumor Necrosis Factor

TPO Thyroid Peroxidase

TSH Thyroid Stimulating Hormone

TSHR TSH Receptor

VIP Vasoactive Intestinal Peptide

CHAPTER ONE

INTRODUCTION

Sleep is the natural state of bodily rest observed in mammals, birds, many reptiles, amphibians and fishes. It is equally a state of unconsciousness from which a person or animals can be aroused. In this state the brain is relatively more responsive to internal than external stimuli. In contrast, coma is also a state of unconsciousness from which a person or animals cannot be aroused (Max, 2006). Sleep is homeostatic; therefore it is controlled by the body’s internal balance (Max 2006). It is considered critical for maintenance of health, support of life, restoration of body and brain functions and promotion of neural-immune interaction (Aurell and Elqvist, 1985; Everson et al., 1989). These are reflected in the roles of sleep in the brain for memory co-ordination and teaching (Turner et al., 2007). Through its role in hormone activities such as in growth hormone, thyroid stimulating hormone and prolactin to mention a few, metabolic processes are properly co-coordinated and carbohydrate storages are carried out (Bonnet and Arand, 2003; Everson and Read, 1995).

Sleep deprivation, a general lack of necessary amount of sleep, which may occur as a result of sleep disorder or deliberate inducement or torture, is deleterious to health when it is prolonged. It has been scientifically observed that prolonged sleep deprivation may result in aching muscles, blurred vision, and clinical depression, and constipation, dark circles under the eyes, daytime drowsiness, and decrease mental activity and concentration, delirium, dizziness, fainting, hallucination, hand tremor, headache, hypertension, irritability, loss of appetite, memory lapses or loss, nausea, nystagmus, pallor, psychosis-like symptoms, severely yawning, sleep paralysis while awake, slowed reaction time, slowed wound healing, synaesthesia, temper tantrum in children, weakened immune system, weight loss, diabetes mellitus type II, obesity without weight gain and death (Gotlieb et al., 2005).

Prion protein pathologies are also associated with alteration in sleep. Rats inoculated with brain homogenates from scrape infected animals demonstrated unusual spiking patterns in the electroencephalogram (E.E.G) about four months after inoculation. During that period slow wave sleep (SWS) and active wakefulness are reduced while drowsiness is increased (Bassant et al., 1984; Bassant et al., 1986). In human, the condition known as fatal familial insomnia is associated with prion disease related to thalamic neurodegeneration (Gibbs et al., 1980). Mutation in prion protein, a glycoprotein on neuronal membrane astrocytes, may underlie the pathological changes that accompany this condition (Monanri et al., 1994). Mice that genetically lack the prion protein gene demonstrated alterations in both sleep and circadian rhythms (Tobler et al., 1997). It has been demonstrated that neuronal cellular prion protein (PrPc) (but not non-neuronal) is involved in sleep homeostasis and sleep continuity (Manuel et al., 2007).

The main systemic disorder resulting from prolonged sleep deprivation in laboratory animals are negative energy balance, low thyroid hormones, and host defense impairment (Bergmann et al., 1989; Everson and Nowak, 2002). Prolactin, a lactating hormone and one of the anabolic hormones involved in sleep promoting activities was observed to be reduced during prolonged sleep deprivation (Vontruer et al., 1996).

Recent finding on the alterations in thyroid hormones in sleep deprived rats point to the brain as the essential site of sleep deprivation effects (Utiger, 1987). The hypothalamus and pituitary are the main sites of hormone production and regulation in the brain. Relatively, little is known regarding other neuro-endocrine consequences of sustained sleep deprivation and whether there is broad pituitary or hypothalamic involvement. It has also become necessary to survey the possibility of changes in the levels of some fertility hormones with sleep deprivation. The hormones of interest here are the follicle-stimulating hormone (FSH), luteinizing hormone (LH), ooestradiol, testosterone, Prolactin and thyroid-stimulating hormone (TSH).

Following the various relationships between sleep deprivation, prion protein (PrP) and hormones, it is necessary to explore the possible changes sleep deprivation may induce on PrP and some fertility hormones.

1.1 Sleep

In animals, sleep is a naturally recurring state characterized by altered consciousness, relatively inhibited sensory activity, and inhibition of nearly all voluntary muscles. It is distinguished from wakefulness by a decreased ability to react to stimuli and it is more easily reversible than being in hibernation or a coma (Macmillian, 1981). Sleep is the natural state of bodily rest observed in mammals including humans. It is also observed in birds, many reptiles, amphibians and fishes. It is common to all mammals and birds. It is equally a state of unconsciousness from which a person or animal can be aroused. In this state, the brain is relatively more responsive to internal than external stimuli. The unconscious state of sleep is distinguished from that of coma by the fact that unconsciousness of coma in mammal or animal cannot be aroused (Max 2006; Ursin, 1983).

1.1.1 Biology of Sleep

Sleep was thought to be a passive state but it is now known to be a dynamic process. It is homeostatic, therefore, it is controlled by the body’s internal balance.The brain is the seat of internal balance and it is active even during sleep. The brain is made up of parts and nerve centres that elicit nerve-signaling chemicals called neurotransmitters. The state of the brain activities during sleep and wakefulness results from activating and inhibiting forces that are generated within the brain. The neurotransmitters such as serotonin and norepinephrine of the brain control whether one sleeps or keeps awake by acting on the nerve cells or neurons in different parts of the brain as the need arises. The frontal lobe of the brain keeps the body awake. It is the centre of planning, the memory search, motor control and reasoning. The thalamus is for attention and sleep. The hypothalamus, located under the thalamus plays the role of promoting the type of sleep called slow wave sleep (SWS). The brain stem plays a great role in sleep and wakefulness. The brain stem is a set of neural structures at the base of the brain. It connects the brain to the spinal cord. It is made up of the medullar, the pons and the reticular formation. While the reticular formation helps to keep, the body awake and alert, the pons is involved in the sleep and control of facial muscles. The neurons at the brain stem actively cause sleep by inhibiting other parts of the brain that keep a person or animal awake (Sherwood, 1997).

1.1.2 Regulation of Sleep

Sleep, one of the most sophisticated integrative functions in higher animals, appears to be regulated by the brain in conjunction with a variety of endogenous humoral factors. These factors are called sleep substances (Inoue, 1985). These substances are endogenous in the brain, cerebrospinal fluid and blood. These substances under the high physiological demand for sleep in the organism are produced in the brain stem and transferred to the whole brain via the body fluid (especially CSF) to induce or maintain sleep. These substances include peptides or protein, hormones and somnogenic (Pappenleiner, 1975; Schoeneberger, 1977).

It is well known that growth hormones (GH) is secreted during delta sleep at first few periods of sleep cycle in humans (Gronfier et al., 1996). It equally plays a part in subsequent appearances of rapid eye movement (REM). Prostaglandin D2 (PGD2) has been revealed as one of the most promising candidates for an endogenous sleep substance. It induces slow wave sleep (SWS) in rats under restrained conditions (Obal, 2003). Adenosine, a purine nucleoside produced during nucleic acid metabolism and protein catabolism builds up in our blood when we are awake. At a level of accumulation, it stimulates drowsiness/sleep and break down gradually when we are asleep to enable restoration of wakefulness (Obal, 2003). Other substances such as emphetamines, caffeine, cocaine and crack cocaine, energy drinks and methylphenidate cause wakefulness (Abaraca et al., 2002). Wakefulness actually refers to a period of consciousness. The term consciousness therefore refers to subjective awareness of private inner world of one’s own mind, that is, awareness of thoughts, dreams and events. Maximum alertness depends on attention and getting sensory impute that energizes the reticular activity system (RAS) of the reticular formation of the brain stem and subsequently the activity level of the central nervous system (CNS) as a whole.

1.1.3 Functions of Sleep

Sleep is considered critical for maintenance of health, support of life, restoration of body and brain functions and production of neural interaction (Aurell and Elqvist, 1985; Everson et al., 1989). These are reflected in the roles of sleep in the brain for memory consolidation and learning. Working memory is important. It keeps information active for further processing and support higher-level cognitive functions such as decision making, reasoning and episodic memory. These functions were shown to be affected by sleep deprivation in humans to a drop of about 38% in comparison to non-sleep deprived individuals (Turners et al., 2007).

Through its roles in hormonal activities, such as growth hormones, thyroid stimulating hormones and prolactin to mention but a few, metabolic processes are properly coordinated and carbohydrate storages are carried out (Bonnet and Arand, 2003; Bergmann et al, 1989 and Everson and Read, 1995). It has been shown that sleep, more specifically slow wave sleep, does affect growth hormone levels in adult men. During eight hours sleep it was found that the men with high percentage of SWS (average 24%) also had low growth hormone secretion while subjects with a low percentage SWS (average 9 %) had high growth hormone secretion. There are multiple arguments supporting the restorative functions of sleep. We are rested after sleeping and it is natural to assume that this is a basic purpose of sleep. The metabolic phase during sleep is anabolic and anabolic hormone such as growth hormones as mentioned earlier are secreted preferentially during sleep (van Cauter et al., 2000).

1.2 Sleep Deprivation

Sleep deprivation is a general lack of the necessary amount of sleep. This may occur as a result of sleep disorders, active choice or deliberate inducement such as interrogation, for purposes of keeping watch for security reasons, prolonged study or research and some times for torture. During sleep deprivation there is a progressive increase in peripheral energy expenditure to nearly double normal levels, resulting to negative energy balance (Everson and Wahr, 1993). In response to this metabolic demand, an increase in serotonin and catecholamines act on both the frontal lobe of the brain stem to keep the body awake (NIH Pub, May 2007).

1.2.1 Sleep Disorders

The actual sleep disorders include sleep apnea (apnoea), narcolepsy, primary insomnia, periodic limb movement disorder, restless leg syndrome and the circadian rhythm sleep disorders. Sleep apnoea is caused by lack of Co2 tension in the blood for stimulation of the respiratory centre which in turn causes failure of the autonomic control of the respiration. This becomes more pronounced during sleep. Narcolepsy is the sudden, repetitive attack of sleep occurring in the daytime, causing diverse clinical conditions. Body pains, illness, stress and drugs can equally cause sleep deprivation during such conditions. Elderly people may loose ability to consolidate sleep due to aging factors.

1.2.2 Sleep Deprivation and Associated Problems

When the body is deprived of sleep for a long time, it elicits a number of negative responses resulting to a number of diseases (Rechtcheffen, 1983). Such responses include negative energy balance, protein malnutrition reduction in circulating anabolic hormones and host defense impairment (Everson, 2004). Though food consumption remained normal in sleep deprived rats fed with a diet of high protein-to-calorie ratio, body weight loss was more than 16% of baseline, development of skin lesions was hastened and longevity was shortened 40% compared with sleep deprived rats fed with the calorie augmented diet. Food consumption of the calorie fed rats was lower during baseline than that of protein fed group but during sleep deprivation increased to amounts 250% of normal without net body weight gain, implying negative energy balance and malnutrition during prolonged sleep deprivation (Everson and Wehr, 1993). The negative energy balance is not due to malabsorption of calorie or diabetes but may be a metabolic response to infectious processes (Everson and Crawley 2004). In a study by Zager and co-workers, rats deprived of sleep for 24 hours were found to have 20% decreases in white blood cell count when compared with the control group (Zager et al., 2007). It was equally shown that in prolonged sleep deprivation or sleep loss, there is a progressive increase in circulating phagocytic cells, mainly neutrophils, migrating into extra vascular liver and lung tissues. These are consitent with tissue injury or infection and are of significant changes in immune system. Also, it was noted that sleep deprivation and strainous exercise result to decrease in neutrophils, monocytes, Eosinophils and lymphocytes. Also major subgroups of immune factors such as CD4, T cells, CD8 T cells, B cells and NK cells were reduced. Furthermore, Cytokines, low molecular weight proteins whose receptors are produced in Central Nervous System (CNS) which mediate many aspects of the host defense, inflammation and tissue remolding and also powerful modulators of sleep-wake behavior are altered during sleep loss in response to microbial infections (Opp and Toth, 2003). The hypothesis that chronic sleep loss impairs immune competence is most strongly supported by observation that chronic sleep deprivation in rats results to intestinal bacterial proliferation, microbial penetration into the lymph nodes, septicemia and eventual death (Opp and Toth, 2003). Conversely, experimental challenges tests have shown that bacterial products and particular immuno modulators such as Cytokines and Chemokines can alter the amount of sleep and its stages (Krueger et al., 2001; Obal and Krueger, 2003). The demonstrated link between cytokines and sleep was the observation that sleep deprivation enhanced the ability of leucocytes antiviral interferon (IFN), which has a role in modulation of sleep. The type I interferon are well known as antiviral cytokines and may be particularly important as modulators of viral induced alterations in sleep. However, both type1 (alpha/bets IFN) and type II (Gamma IFN or immunocytes) are known to modulate sleep. Also influenza, immune deficiency viruses (HIV, in human, FIV in cat and NDV in mice), all induce sleep alteration (Opp and Toth, 2003; Norman et al., 1990).

1.2.3 Sleep Deprivation and Protein Metabolism

Protein malnutrition and malformation are part of the negative effects elicited by prolonged sleep deprivation. Sleep is associated with increased protein synthesis in several brain regions as well as the whole cerebrum (Ramm and Smith 1990). Sleep deprivation on the contrary reduces the level of certain proteins in the rats basal forebrain and hippocampus (Basheer et al., 2005). As stated earlier, sleep deprivation affects various aspects of protein including metabolism and translational changes involving unfolding and misfolding of proteins (Schroder and Kaufmann, 2005; CiIrelli et al.,2006).

However, sleep deprivation promotes endoplasmic reticulum stress hormones and production of eif2 and membrane proteins (Proud, 2005). All components of unfolded protein response (UPR) or endoplasmic response (ER) stress were found after 6 hours of sleep deprivation in mouse neocortex, including increase in P-eif2α as well as free BIP, GRP78 and phosphrylated protein kinase-like ER kinase (PERK), a key kinase that phosphorylates eiF2α (Naidoo et al., 2005). During prolonged sleep deprivation, further changes such as transcript coding for several immunoglobulins, stress response protein such as macrophage inhibitor factor-related protein 14, heat-shock protein 27, alpha-β-crystallin and minoxidil sulfotransferase, globins and cortistatin are observed. At molecular level also several plasticity-related genes were strongly induced after acute sleep deprivation only and several glial genes were down regulated in both acute and long-term sleep deprivation conditions but to different extents. These findings suggest that sustained sleep loss may trigger a generalized inflammatory and stress response in the brain (Cirelli et al., 2006). It has equally been identified that endoplasmic reticulum(ER) resident chaperon, immunoglobulin binding protein (BIP) increase with sleep deprivation. The endoplasmic reticulum is the key cellular marker and master regulator of signaling path way called ER stress response or unfolded protein response (Naidoo et al., 2005).

1.2.4 Sleep Deprivation and Prion Protein

Prion protein related pathologies, which are associated with protein misfolding and neurodegenerative disease of the brain, are also associated with alteration in sleep (Gibbs et al., 1980; Monari et al., 1994). Rats inoculated with brain homogenates from scrapie-infected animals demonstrated unusual spiking patterns in the electroencephalogram(EEG) about four months after inoculation. During those periods, slow wave sleep (SWS) and active wakefulness are reduced while drowsiness is increased (Bassant et al., 1984; Bassant et al., 1986). Cats inoculated intracerebrally with brain homogenates from a human infected with Creutzfeldt Jacobs disease, demonstrated increased SWS time, reduce wakefulness and abnormal EEG after 20 minutes of inoculation. In human, the condition known as fatal familial insomnia is associated with prion disease related to thalamic neurodegeneration (Gibbs et al., 1980; Monari et al., 1994). Mutation in prion protein, a glycoprotein on neuronal membrane astrocytes, may underlie the pathological changes that accompany this condition (Monari et al., 1994). Mice that genetically lack the prion protein gene, demonstrate alterations in both sleep and circadian rhythms (Tobler et al, 1997). PrP-null mice have a low sleep pressure, leading to more frequent interruptions of sleep and reduced SWS (Tobler et al., 1997). In other words, the PrP-null mice (PrP %) show longer sleep fragmentation together with an increase of slow wave activity (SWA) during NREM sleep after a short period of sleep deprivation. It has been demonstrated that neuronal cellular prion protein (PrPc) but not the non-neuronal, is involved in sleep homeostasis and sleep continuity (Manuel et al., 2007; Tobler et al., 1997).

1.3 Prion Protein (PrP)

Prion protein is a special type of protein that is present in al mammals. It is encoded by a sinc gene at chromosome 20 (Dickson et al., 1968). Prion protein is expressed predominantly in the brain, spinal cord and lymphoid tissues (spleen, lymp nodes and thymus) (Chiol et al., 2006). The protein can also be found in decreasing amounts in salivary glands, lungs, intestines, liver, kidneys uterus and blood (Eklund et al., 1967). Prion protein is a cell surface protein, anchored by a glycosylphosphatidylinostol anchor (GPI) (Oesch et al., 1985). Prion protein can be found in its natural or normal state referred to as cellular prion protein and designated as PrPc. This cellular prion protein (PrPc) is readily digested by proteinase K, just like other common proteins. Owing to its sensitivity to proteinase k. it is also designated PrPsen. The cellular prion protein (PrPc) can be transformed to abnormal form called prions. Prions are resistant to proteinase k digestion and are therefore designated as PrPres. These prions (PrPres) are the only proteinacious particles that cause disease in vertebrates (Chesebro, 1990).

Prion protein has three-dimensional structure like other proteins. It has highly positively charged N-terminal segment. The N-terminal segment comprising residues 23 – 125 of the protein is flexibly disordered. The N-terminal segment contains four octapeptide repeats, PHGG (G/S) WGQ (between residues 60-93) and a homologous sequence lacking a histidine residue PQG G WGQ (Between residue 52 and 60). It equally has globular fragment. The globular C. terminal fragment 121-231 contains three α- helices and two β-strands (Riek et al., 1996). The hydrophilicity and charge distribution make the first prion protein α-helices unique among all naturally occurring alpha helices (Morrissey and Shakhnovich 1999). Prion proteins have electrostatic interaction and salt bridges stimulated by molecular dynamics. The electrostatic interaction in general and salt bridges in particular plays an important role in prion protein stability. This protein is coded in all mammals by a sinc gene (Dickson et al., 1968). In man, it is transcribed by a sinc gene present on chromosome 20. The molecular structure of prion protein is dictated by the prion gene, the human form of which is abbreviated as PrnP. PrnP encodes for a protein of 254 amino acids in length. PrnP undergoes post-translational modification in two important ways, cleavage and glycosylation. The glycosylation is at two sites. In hamster proteins they are at position 183 and 197.

Prion protein is a cell surface protein anchored by a glycosylphosphatidylinositol anchor (GPI). This is anchored to sphingolipid Rafts (membrane Organisation of GPI-APS into a laterally organized cholesterol sphingolipid ordered membrane domain). From this sphingolipid raft it can be endocytosed by a copper 2 ion activated mechanism (Oesch et al., 1985; Brown, 2001). Following the cleavage of a 22 amino acid signal peptide, mammalian cellular prion protein (PrPc) is exported to the cell surface as N-glycosylated Glycosylphosphatidylinositol (GPI) anchored protein of 208-209 amino acids, with its three dimensional structure retained (Calzolai et al., 2005; Riek et al., 1996; Zahn et al., 2000). PrPc contains an NH2-terminal flexible and random coil sequence of 100 amino acids and COOH- terminal globular domain of about 100 amino acids. The globular domain of the human PrPc is arranged in three α helices corresponding to amino acid 144-154, 173- 194 and 200-228, interspaced with an antiparallel β-pleated sheet formed β- strands at residue 128-131 and 161-164. A single disulfide bond is found between Cysteine residues 179 and 214. The NH2-terminal flexible tail comprises approximately residues 23-124, and a short flexible COOH- terminal domain corresponding to residue 229-230. The DNAs of both hamster and mouse PrP encodes for polypeptides of 254 amino acids (Locht et al., 1986). However, an N-terminal signal peptide of 22 amino acids is removed from these molecules during biosynthesis (Hope et al., 1986; Bolton et al., 1987) and an additional 23 amino acids are removed from the C-terminal of the proteins during glycosylphosphatidylinositol (GPI) addition at Ser 231 (Stahl et al., 1990a), resulting to a mature PrP polypeptide of 210 residues. A single disulfide bond in PrP forms a loop (Turk et al., 1988), which contains two consensus sites for Asn-linked glycosylation at residues 181 and 197. Addition of glycans at these sites generate three main glycosylated and fully glycosylated PrP. High mannose glycans added to the protein in the endoplasmic reticulum are converted to complex or hybrid glycans in the golgi apparatus . PriPsen on the cell surface has a metabolic half-life of 3-6 hours (Borchelt et al,

1990) and most PrP appear to be degraded in non-acidic compartments bound by cholesterol-rich membrane.

Studies on endocytosis of PrP sen indicate that it cycles between the cell surface. Both sulfate glycans and copper have been shown to stimulate the endocytotic compartment with transit time of 60 minutes endocytosis process (Pauly and Harris, 1998). However, the exact mechanism of internalization has been controversial. The GPI anchors of PrPc determines its route (Taraboulos et al, 1995)

Endocytosis remains the process in which materials (in this case, protein) enter the cell without passing through the cell membrane. The membrane folds around the protein outside the cell resulting to the formation of a sack like vesicle into which the material (Protein) is incorporated in this way. A macromolecule such as protein is said to be internalized in the existing component of cells. Numerous mammalian proteins have a special post-translational modification at their carboxy-terminal known as the glycosylphosphatidylinsitol (GPI) anchor, which serves to attach the proteins to the extra cellular leaflet of the cell membrane. The GPI anchor consists of a phosphatidylinsitol group attached to a carbohydrate moiety (trimanosyl-nonactylated glucosamine), which in turn is linked through a phosphodiester bond to carboxy-terminal amino acid of the mature protein. Glycosylphosphosphatidylinsitol anchored proteins (GPI-APS) therefore, represents an interesting amalgamation of the three basic kinds of cellular macromolecules, viz, proteins, carbohydrates and lipids. For prion proteins, the cell surfaces were shown to trafic through the endocytic intermediates and this step was even shown to be necessary for conversion of PrPc to PrPsc. Clathrin coated pits were shown to be instrumental to the endocytosis of PrP. Through this process of endocytosis and internalization, molecular materials are attached to the structures of prion protein.Molecular dynamic stimulation suggest that some attached N-glycans may modulate PrPc stability (DeMarco and Daggett 2005; Ermonval et al., 2003).

Cellular prion protein (PrPc) can be liberated from the cell surface invitro by enzyme phosphoinositol phospholipase (PiPl), which usually cleave the phosphatidylinostol glycolipid anchor (Weisman, 1999). PrPc is readily digested by proteinase K, it is designated with the term PrPsen. Full-length recombinant proteins and as prion protein (amino acid residue 23-231 is denatured by neutral salts such as sulfate and flouride salts, contrary to the report that structure of protein, either basic or acidic are stabilized against denaturation by certain neutral salts such as sulfate and fluoride (Nishimura et al, 2002). Under identical concentration of neutral salts, the structure of sheep prion protein which contains a greater number of glycine groups in N-terminal unsaturated segment than mouse PrP becomes more stabilized. Also in contrast to full-length protein, the C-terminal 121-231 prion protein fragment consisting of all the structural elements of the protein i.e. three α-helices and two short β-strands is stabilized against denaturation by neutral salts. Prion protein has a preferential interaction with glycine residues in the N-terminal segment, consistent with α-helix I. The prion α-helix I is the most soluble of all the prion α-helices reported so far in literature. Increasing the concentration of anions on the prion protein surface perturbs the solubility of the α-helix I, thereby making structural conversion of protein structure to β-pleated sheet (insoluble) by anionic nucleic acid. It is equally reported that DNA can also modulate the aggregating properties of prion protein (Cordeiro et al., 2001). Interaction between prion protein and nucleic acid also leads to the demonstration that prion protein can play a role in nucleic acid metabolism (Gabus et al., 2001). PrPc has a molecular weight of 35-36 KDA and very hydrophilic alpha helical structure. It can pass through the filter paper with an average pore diameter of 20-100nm, suggesting a size range consistent with conventional viruses (Eklund et al., 1963). It has sedimentation constant ranging from 200 S to 2000 S. (Prusiner et al., 1977). Prion protein is highly expressed within the nervous system, although its content varies among distinct brain regions. It is predominantly expressed in the brain, spinal cord and lymphoid tissues (spleen, lymph nodes and thymus) (Chio et al., 2006). The protein can also be expressed in decreasing amount in various components of immune system, salivary glands, lungs, bone marrow, blood, intestine, liver, kidney, uterus and peripheral tissues (Eklund et al., 1967). The expression of PrPc by neurons within the central nervous system is particularly in the hippocampus, neocortex, spinal motor neuron, and cerebella, purkinje cells (Piccardo et al., 1990; Sales et al., 1998). Modest amounts of PrPc are also expressed in glial cells within the brain and spinal cord, in peripheral tissues and in human T-cells. B-cells, monocytes and dendrites cells but not as much in blood cell. Immunoblotting studies revealed that PrPc glycoforms and the composition of N-linked glycans or PrPc in human peripheral blood mononuclear cells are different from those of the brain or neuroblastoma cells (Ruliang et al., 2001).

1.3.1 Functions of Prion Protein

Nature has roles or functions vested on the cellular prion protein for their expressions on various cells, tissues and organs of the humans and other mammals. Although, some of these functions are not yet clearly elucidated, some experimental demonstrations are evident.

1.3.2 Prion Protein and Cell Membrane Viability

The widely reported and accepted theory that cellular prion protein (PrPc) is anchored to the cell surface and invariably on the neuronal surface by glycosylphosphatidylinositol, suggests a role in the cell signaling or adhesion. It is reported that the hippocampal slices from the PrP null mice have weakened GABAA (γ-aminobutyric acidA) receptor-mediated fast inhibition and impaired long term potentiation. This impaired synaptic inhibition may be involved in the epileptiform activity seen in Creutzfeldt Jakob disease. Therefore it is argued that loss of function of PrPc on the neuronal surface may contribute to the early synaptic loss and neuronal degeneration (Oesch et al., 1985; John-collinge et al., 1994).

1.3.3 Prion Protein and Sleep

In an experimental design, it was demonstrated that after sleep deprivation PrP null mice showed a larger degree of sleep fragmentation and latency to enter rapid eye movement sleep and non rapid eye movement. Also during sleep recovery experiment the amount of NREM sleep and the SWS were reduced in PrP null mice. This finding demonstrated that neuronal PrPc is involved in sleep homeostasis and sleep continuity while non-neuronal PrPc is not involved (Manuel et al., 2007).

Altered sleep pattern and circadian activity rhythm have been observed in mice devoid of PrP (Tobler et al., 1997). There are some evidences that fatal familial insomnia, an inherited human prion disease results from deficiency of normal prion protein, a deficiency that occurs because mutant prions are unable to fulfill normal functions. PrPc null mice were observed to develop normally at early stage of life but underwent severe ataxia and purkinje cell degeneration at advanced ages (Manuel et al., 2007). Impaired coordination observed in aged PrP null mice (70 weeks and above) was attributed to lack of PrPc and correlated with the loss of cerebella purkinje cells in PrP null mice. Purkinje cells survive longer with the presence of cellular prion protein (Katamine et al., 1998). They showed a slight increase in locomotor activity during exploration of environment. Also under acute stress, such as restraint or electric footshock, mice lacking PrP showed reduced levels of anxiety when compared to the PrP expressed mice. Anxiety is accompanied by a characteristics set of behavioural and physiological responses that tend to protect the individual from danger and is taken as part of a universal mechanism of adaptation to adverse condition (Chen et al., 1995).

1.3.4 Anti-Apoptotic Function

Cellular prion protein has anti-apoptotic effects. It plays a role against Bax-mediated neuronal apoptosis. Bax-mediated apoptosis refers to the Bcl-2 associated protein-X (Bax) mediation apoptosis. PrPc potently inhibits Bax-induced cell death in human neurons. Deletion of four octapeptide repeat of PrPc by mutation or otherwise completely or partially eliminates the neuroprotective effects of PrPc. PrPc remains anti-apoptotic despite truncation of glycosylphosphatidylinositol anchor signal peptide, indicating that neuroprotective form of normal prion protein does not require the abundant cell surface GPI anchored PrP (Bounher et al., 2001; Kuwahara et al., 1999). It was also reported that neuronal PrPc engagement with stress-inducible protein-1 and laminin plays a key role in cell survival and differentiation. This was demonstrated from the PrPc expression in astrocytes (Star shaped glial cells in the brain and spinal cord).The study evaluated whether PrPc expression in astrocytes modulates neuron-glia cross-talk that underlies neuronal survival and differentiation. Astrocytes from wild-type mice promoted a higher level neuritogenesis than astrocytes obtained from PrPc null animals. Remarkably, neuritogenesis was greatly diminished in co-cultures combining PrPc null astrocytes and neurons. Laminins (LN) (Major proteins in the basal lamina, a protein network foundation for most cells and organs) hold cells and tissues together.They are secreted and deposited at the extracellular matrix by wild type astrocytes; presented a fibrillary pattern and was permissive for neuritogenesis. Conversely, laminin coming from PrPc null astrocytes displayed a punctuate distribution, and it did not support neuronal differentiation.

Additionally, secreted soluble factors from PrPc-null astrocytes promoted lower levels of neuronal survival than those secreted by wild type astrocytes. PrPc and stress-inducible protein-1 were characterized as soluble molecules secreted by astrocytes which participate in neuronal survival. Taken together, these data indicate that PrPc expression in astrocytes is critical for sustaining cell to cell interactions, the organization of extracellular matrix, and the secretion of soluble factors, all of which are essential events for neuronal differentiation and survival (Lima et al., 2007). PrPc also interact with laminin for its function of memory processing, consolidation/retention and cognitive performance in mammals, especially humans. It was demonstrated that hippocampal PrPc plays a critical role in memory processing through interaction with Laminin. One of the plausible hypotheses is based on the interaction of laminin with tissue type plasminogen activator/plasmin proteolytic cascade. On the other hand, laminin stimulates neurite outgrowth, and the most abundant laminin isoform in the hippocampus is LN10 (α5β1γ1), which is produced and secreted by neurons. These cells bind to LN10 through integrin α3β, as well as through PrPc. The PrPc binding domain maps to the COOH-terminal domain of lamininγ-1 chain, and only PrPc binds to this domain,through which it is able to promote neurite outgrowth (Indyk et al., 2003). PrPc interaction with laminin is also involved in the neuronal signaling process and signal transduction in neuronal cells (Spielhaupter and Schatzl, 2001).

It has been reported that PrPc plays a key role in maintaining myelin, a fatty substance that forms a sheet around nerves and helps transmit nerve signals. It was also found that mice without PrP in certain nerve cells suffer from a demyelinating disease that closely resembles one seen in humans. A published paper which suggested that mice without PrP had damage to their peripheral nerves, triggered a scientific probe on this report. A team of scientists examined five strains of mice lacking the PrP gene and found that all showed this peripheral nerve damage by ten weeks of age. Since this finding did not actually answer what was behind the nerve damage, a one- year old mice was studied and found that their sciatic nerve (the large nerve in the back that runs into the legs) had lost myelin. Then mice that lacked PrP in some cells but not in the others were studied to see which cells were behind the demyelination. The result was a surprise. When PrP was present on the axons (the fibers that conduct electrical impulses), it prevented disease.

When it was lacking in axons but present in the so called Schwarnn cells that actually form the myelin sheet, the mice got sick. Though the Schwarnn cell are the ones affected when PrP is missing, the protein must be present in axons to prevent disease (Radovanovic et al., 2005).

1.3.5 Protein and Immune System

Despite the involvement of specific immune cell-type in the accumulation of PrPsc in peripheral lymphoid compartments at early stages of prion disease, no attention has been paid to whether PrPc is depleted in the immune cells and possible consequences of immune responses. Some data show that PrPc may play important roles in the development and maintenance of immune system, as well as in specific cellular immunological responses (Aguzzi et al., 2003). Studies also suggest that PrP plays a role in the cultivation of lymphocytes (Li et al., 2001). As noted earlier, the cellular prion protein (PrPc) is expressed widely in immune system in haematopoietic stem cells and mature lymphoid and myeloid compartment in addition to cells of the central nervous system. It is up regulated in T-cells activation and may be expressed at higher levels by specialized classes of lymphocytes. Furthermore, antibody cross-linking of surface PrP modulates and T-cell activation, leads to the rearrangements of lipids raft constituents and increased phosphorylation of signaling proteins. These findings appear to indicate an important, but, as yet, ill-defined role of PrPc in T-cell. Although, PrP mice has be been reported to have only minor alterations in immune function, recent work has suggested that PrP is required for self renewal of haematopoietic stem cell (Aguzzi et al., 2003; Choi et al., 2005).

1.3.6 Prion Protein and Muscular Tone

Prion protein (PrPc) has roles or functions beyond the nervous and immune systems. Expression of PrPc is increased in sporadic and hereditary inclusion, body myositis and myopathy, polydermatomyositis, and neurogenic muscles atrophy. A uniform pattern of increased PrPc expression was described in a series of muscular disorders. Interestingly, both glycoform profile and size of PrPc in normal muscle are distinct from human brain (Kovacs et al., 2004). Based on these findings, it was suggested that PrPc may have a general stress-response effect in neuromuscular disorders (Kovacs et al., 2004). This hypothesis is supported by accumulation of PrPc in muscle fibers of an experimental model of chloroquine- induced myopathy (Furukawa et al., 2004). In addition, PrPc was up regulated when myotubes differentiate from immortalized C2C1 murine myoblasts (Brown et al., 1998). PrPc content progressively increased during maturation of myocytes in primary culture of skeletal muscle, attributed to both transcriptional and post translational changes. Fast muscle fibers present a higher concentration of PrPc than slow fibers and are consistent with a role of PrPc in skeletal muscles physiology. A severe dilated cardiomyopathy has also been described in patients diagnosed as sporadic CJD, and a heart biopsy contained evidence of the presence of PrPsc. Since no other cause was found, it was suggested that the disease is derived from accumulation of PrPsc into the heart (Ashwath et al., 2005). Recently disease associated PrP was also detected in cardiac myocytes of elk and whitetail deer infected with chronic wasting disease(CWD),but the heart physiology was not evaluated (Jawell, et al, 2006). These data raised the thought that PrPc may have important functions in both skeletal and cardiac muscles.

1.3.7 Abnormal Prion Protein

The cellular prion protein (PrPc) can be transformed into abnormal forms. The abnormal forms of prion protein can be referred to as prions. Prions or the abnormal form of prion protein consist of the only proteinacious infectious particles that causes diseases in vertebrates (Chesebro, 1999). Prion is equally a small infectious pathogen containing protein but apparently lacking nucleic acid. The prion protein is the critical component of this infectious pathogen or agent and may infact be the exclusive constituent. Prion is the only protein with the ability to transmit biological information through the propagation of alternative protein folding without changes in the genome (Upstair and Lindguist, 2002). Prions are resistant to proteolytic action of proteinase K amd are designated as PrPres. Prion appears to be crystalike clusters of PrP molecules that can grab normal and soluble PrP molecule and convert them to a solid and insoluble crystal like state.

The word prion was coined in 1982 by Stanley B Prusiner, from a portmanteau derived from the word protein and infection (Prusiner, 1982). Radiologist Tikvah Alper and Mathematician John Stanley Griffith developed the hypothesis during the 1960s that some transmissible spongiform encephalopathies are caused by infectious agent consisting solely of protein (Alper et al., 1997). Their theory was developed to explain the discovery that the purported mysterious infectious agent causing the disease scrapie and Creutzfeldt-Jacob disease resisted ultraviolet radiation. Stanley B Prusiner of the University of California, San Francisco announced in 1982 that his team had purified the hypothetical infectious prion and that the infectious agent consisted mainly of a specific protein. Prusiner coined the word “prion” as a name for the infectious agent while the specific protein that the prion was composed of is known as prion protein (PrP) (Gary, 1986). Prion protein may occur both in infectious and non-infectious forms.

Proteins which are linear chains of small molecules called amino acids, fold into complex three dimensional shapes to carry out their functions. Prion protein some times folds into the wrong shape. The misfolded copies of the protein have been found to accumulate in the brain to cause a number of brain disease.

1.3.8 The Pathogenicity of Prion

Unlike the normal prion protein (PrPc) which does not cause any harm, the misfolded prion protein (PrPres) cause neurological diseases. In the brain large deposits of this misfolded protein are found in form of plaques which are believed to be attempt by the brain to detoxify the infectivity of PrPres. Infectivity relates to particle size. Small prions are much more efficiently infectious than large ones, yet there is a lower size limit, below which infectivity is lost. As the particle size is increased from single molecules to particles containing thousands of molecules, there is a sudden jump in the infectivity once you get to a minimum infectious particle size of at least six PrPres molecules per particle. Soon the most infectious particles appear (Equivalent weight of 14-28 PrPres molecules per particle) (Science Daily, 2004; 2005).

The formation and accumulation of PrPres are made possible by the misfolding copies attaching to the cellular prion protein (PrPc) and acting as templates for continual misfolding and accumulation of PrPres. The binding of the misfolded or abnormal prion protein (PrPres) to the normal prion protein (PrPc) catalyses conformational change from PrPc to PrPres. This reaction is assisted by a hypothetical specie specific factor termed “Protein X”. All these follow a theoretical heterodimer model (Gauljduset, 1988; Lansburg and Caughey, 1995) and the nucleation (Seed) dependent polymerization model (Gajdusek, 1988; Jarrett and Lanbury, 1993). The heterodimer model proposes that PrPres exists in a stable monomeric state that can bind PrPc, forming a heterodimer and catalyse a conformational change in PrPc to form a homodimer of PrPres. The PrPres homodimer then dissociates to give two PrPres monomers. Fundamental aspects of this model are that PrPres is more stable thermodynamically than PrPc.Conversion of PrPc to PrPres is rare unless catalysed by a pre-existing PrPres template, and the PrPres homodimer tends to dissociate into monomers. According to the model, this process also requires the assistance of a hypothetical, species- specific factor termed ‘protein x’ (Jarret and Lansbury, 1993).

1.3.9 The Diseases of Prion (PrPres)

Following the various processes or mechanisms discussed above, the conformational changes of PrPc to PrPres remains stable in their misfolded states. The accumulation of this abnormal form of prion protein (PrPres) in form of plaques results to a number of neurodegenerative diseases depending on how and where the intensity of the accumulation occurred. The diseases caused by the accumulation of prion or the abnormal prion protein (PrPres) have been generally identified as and called Transmissible Spongiform Encephalopathies (TSE) (Chesebro, 1999; Gibs et al., 1980). These unusual groups of neurodegenerative diseases can be transmitted between individuals by either inoculation or ingestion of diseased brain or other tissues and by genetic mutation (Gibbs et al., 1980). TSE can be seen or identified in various forms: as Scrapie in sheep, Bovine spongiform Encephalopathy (BSE) in cattle, and Creutzfeldt-Jakob Disease (CJD), Kuru, Gerstmann Straussler-Scheinker Syndrome (GSSS), and Iatrogenic TSE in humans. Also identified among these TSE disease include chronic wasting diseases (CWD), familial Creutzfeldt-Jakob disease (fCJD), variant CJD (vCJD), Fatal familial insomnia (FFI) and Transmissible Mink Encephalopathy (TME). In addition to its presence in sheep, cattle and humans, TSE occur in many other different animal species including nonhuman primates, mice, hamsters, rats, guinea pigs, mink, goat, pigs, elk, deer, cats and a variety of exotic felines and bovids in zoos (Chesebro, 1990).

The Transmissible Spongiform Encephalopathy became recognized as disease in sheep (Scrapie) in Europe over 200 years ago. Sheep breeders became aware that scrapie free flocks developed disease after introduction of new stock from the infected flocks. This suggested that the disease might be transmissible. Experimental transmission was reported as early as 1899 by Bonnoitt. However, the six months incubation period observed suggested that these sheep might have been naturally infected prior to introduction. Tr The mechanism of natural transmission of scrapie remains uncertain. Placenta and other tissue can contaminate pastures at the time of birth, and uninfected flocks have developed the disease when maintained on such pastures without any direct contact with infected sheep. This would appear to explain the finding in Iceland that scrapie-free sheep became infected when introduced 3 years after eradication of infected flocks (Gajdusek, 1996). Sheep scrapie provides an unusual opportunity to compare natural and experimental TSE disease processes. Although there are no known genetic cases of TSE disease in animal comparable to those seen in humans, allelic variations in the sheep PrP sequence influences susceptibility to both natural and experimental scrapie infection (Bossers et al., 1997).

Bovine Spongiform Encephalopathy is the TSE disease found in cattles. Since 1986, the Bovine spongiform Encephalopathy (BSE) epidemic in the United kingdom has focused international attention on the TSE family of diseases. The origin of BSE is not clear. It may have been derived from an unusual strain of sheep scrapie (Hope et al., 1999), or it might represent a cattle TSE disease present at such a low levels as to have escaped detection previously. However, several Laboratory tests have identified similarities in BSE from sources tested in contrast to most commonly known isolates of feeding of protein supplements contaminated with the rendered tissue of BSE positive cattle. Changes in the rendering process such as exchanging of fat extraction by organic solvents and switching from batch heating to continuous flow heating apparently led to the survival of sufficiently infectivity in the final meat and bone meal product to allow transmission by feeding this material to other cattles. BSE has also been transmitted to other species by feeding of contaminated meat and bones meat to ungulates and large felines in zoos and probably also domestic cats. Transmission to man has also been suggested by appearance of variant Creutzfeldt Jakob disease (vCJD) in small group of younger humans, primarily in the United Kingdom. In cattles the age of onset of BSE is usually 2-5 years. The disease predominantly affects dairy cattles, presumably because these cattles are fed more of the contaminated high protein supplements than are other classes of cattles. Also these animals are usually maintained in production longer than are beef cattles, leading to greater chances of clinical disease. The predominant clinical signs are gait ataxia and changes in behavior or personality, such as aggressiveness or wariness. Infectivity and PrPres are found almost exclusively in the nervous system. The spleen and lymphoid tissues appear to be involved to a much lower extent than in sheep scrapie or other TSE disease models. At present the investigation or test for brain PrPres could be done with Western blotting, enzyme linked immunosorbent assay or immunochemistry. In contrast to BSE which is transmitted to man by possible consumption of infected beef or bovine products, there is no diagnostic evidence of spread of sheep scrapie to humans. Therefore, there may be important fundamental differences between scrapie and BSE in their interaction with different hosts (Caughey and Chesebro, 1997).

Chronic Wasting Disease (CWD) is the TSE in deer and Rocky Mountain elk in Colorado, Wyoming, Nebraska, and Montana. It is another example of TSE disease of unknown origin. Its spread appears to be enhanced by the abnormal population densities found in such facilities as “game farms” though the actual mechanism of transmission is not known (Miller et al, 1998). CWD is found in wild ruminants on the same range as domestic cattles and this raises concern that CWD could be transmitted to cattles and possibly might also pose a risk for human infection similar to BSE.

‘Kuru’ is a TSE disease found in the Eastern Highlands of Papua New Guinea around 1950s. It was discovered that due to religious reasons, the people ingested the brain tissues of dead relatives and through this, the disease spread. The common symptoms of TSE are seen and at terminal stages the patients are usually mute, rigid and unresponsive (akinetic mutism) with decorticate or decerebrate posture as well as fecal and urinary incontinence.

Fatal familial insomnia is another human transmissible spongiform encephalopathy that is deadly. It is a very rare autosomal dorminant inherited prion disease of the brain. It is almost always caused by a point mutation in PrP Condon 178. This results in the substitution of asparagines for aspartic acid. It can also develop spontaneously in patients without inherited mutation in a variant called sporadic fatal insomnia (SFI). This TSE disease is characterized clinically by dysautonomia, dementia, hallucination, panic attack, phobia, motor signs. Also other symptoms such as profuse sweating, pinprick pupils surden entrance into menopause for women and impotence from men, neck stiffness, elevated blood pressure and heart rate. FFI is characterized pathologically by severe atrophy of the anterior ventral and mediodorsal thalamic nuclei. The age onset of this disease is variable, ranging from 30-60 years, with an average of 50 years. However, the disease tends to prominently occur in later years. Death usually occurs between 7 and 36 months from onset. The presentation of disease varies considerably from person, even among patients from within the same family (Max, 2006, Random House, 2006). Since point mutation in codon 178 of cellular prion protein (PrPc) is related to fatal familial insomnia, it therefore buttresses the earlier statement that normal prion protein enhances sleep processes or ability to maintain normal sleep. It was noted that the main systemic disorders resulting from prolonged sleep deprivation in laboratory animals are negative energy balance, low thyroid hormones and host defense impairment (Bergman et al., 1989; Everson and Nowak, 2002). Also Prolactin, a lactating hormone and one of the anabolic hormones involved in sleep promoting activities was observed to be reduced during prolonged sleep deprivation (Obal et al., 1997; Zhang et al., 2001).

1.4 Endocrine System

The endocrine system is a system of glands, each of which secrets a type of hormone into the blood stream to regulate the body functions. The endocrine system is information signal system like the nervous system. Hormones regulate many functions of an organism including mood, growth and development, tissue function and metabolism. The endocrine system is made up of series of ductless glands that produce and secrete hormones. When a number of glands signal each other in sequence, they are referred to as axis. An example is the hypothalamic pituitary-adrenal (HPA) axis. Typical endocrine glands are the pituitary, thyroid, and the adrenal glands. Endocrine glands have general features of ductless nature, presence of vascularity and intracellular vacuoles or granules where their hormones are stored. In contrast, exocrine glands such as salivary glands, sweat glands, and glands within the gastrointestinal tract, tend to be less vascular and have ducts or hollow lumen. In addition to the endocrine organs mentioned above, many other organs that are part of other body systems, such as the kidney, liver, heart and gonads have secondary endocrine functions. The kidney for instance, secretes endocrine hormones such as erythropoietin and renin.

Endocrine hormones are secreted directly into the bloodstream while the exocrine hormones are secreted into ducts from where they flow from cell to cell by diffusion. The exocrine hormones diffuse from cell to cell by a process known as parachute signaling. All hormonal signals involve biosynthesis of a particular hormone in a particular tissue, storage and secretion of the hormones, transport of the hormone to the target cells, recognition of the hormone by an associated cell membrane or intracellular receptor protein, relay and amplification of the received hormonal signal transduction process. This leads to a cellular response. The reaction of the target cell may be recognized by the original hormone producing cells, leading to a down–regulations in hormone production. This down-regulation is known as homeostatic negative feedback loop. The human endocrine system consists of several integrated systems that operate via feedback loops. Several important feed back systems are mediated via the hypothalamus and the pituitary (Sherwood, 1997). These important regulation via feedback loops include the regulation of triiodothyronine/thyroxine (T3/T4) production/activities through the thyroid releasing hormone-thyroid stimulating hormones- T3/T4 loop; the regulation of sex hormones production/activities through the Gonadotropin releasing hormones-Luteinizing/Follicle stimulating hormone(LH/FSH)-sex hormones loop; corticosteroid releasing hormone-Adrenocorticosteroid hormone-Cortisol loop, for production of cortisol; and the renin Angiotensin-Aldosterone loop for production of aldosterone (Sherwood, 1997).

Chronic sleep loss can reduce the capacity of even young adults to perform basic metabolic functions such as processing and storing of carbohydrate or regulation of hormone secretion of the endocrine system. It was discovered on the 6th day of sleep deprivation on young men, that there were profound alterations of patients with type 2-diabetes. Their ability to secrete insulin and to respond to insulin both decreased by about 30%. Sleep deprivation also altered the production and actions of the other hormones, such as dampening the secretion of thyroid stimulating hormones and increasing the blood level of cortisol, especially during the afternoon and evening. It was equally found that the metabolic and endocrine changes resulting from a significant sleep debt mimic many of the hallmarks of aging. Sleep loss induces excessive cortisol secretion, increased β-endorphin level, and altered physiological and anatomical changes such as increased metabolism and adrenal hypertrophy (Bruce et al., 1986). In another incidence,higher serum levels of cortisol, growth hormone and testosterone were discovered during 5 days sleep deprivation and physical strain while such difference were not found in catecholamines, androsterone, dihydrotestosterone, luteinizing hormone,triiodothyronine and thyroxine (Opstad and Aakvaag, 1982).

Prolonged sleep deprivation in rat results in augmented sexual activity. These behavioural effects seem to be in contradiction to the fact that stress in general inhibits the hypothalamic-pituitary-gonadal axis. The CRH, GC and β-endophin inhibit gonadotropin-releasing hormones and testosterone. In fact, four days of REM sleep deprivation in rats culminates in low levels of testosterone, estrone and oestradiol, but high levels of progesterone and corticosterone. Despite the lowered levels of testosterone, in sleep deprived male rats, there is a marked increase in genital reflexes indicated by penile erection and ejaculation, both in young and aged rats. Castrated REM sleep deprived rats treated with progesterone, but not with testosterone display more genital reflexes than vehicle treated rats. To restore male sexual behavior that includes mounting, intromission and ejaculation in REM sleep deprived rats, testosterone is essential. Thus, it seems that progesterone is vital to produce the full range of male reproductive behavior (Anderson et al., 2004; Cardinali and Pandi-Perumal, 2006).

Research findings on the alterations in thyroid hormones in sleep deprivation in rats point to the brain as the essential site of sleep deprivation effects (Utiger, 1987). The hypothalamus and the pituitary are the main sites of hormone production and regulation in the brain. Relatively more is needed to be known regarding other neuroendocrine consequences of sustained sleep deprivation and whether there is broad pituitary or hypothalamic involvement. Subsequently, it has also become necessary to survey the possibility and extent of changes in levels of some main fertility hormones during prolonged sleep deprivation knowing that sleep deprivation to some extent affects the Hypothalamic-pituitary-Adrenal axis (Meelo et al., 2002). The hormones of interest here are the Follicle stimulating hormones (FSH), luteinizing hormones (LH), prolactin, oestradiol in females and Testosterone in males and the Thyroid stimulating hormone (TSH).

1.5 Hormones

Generally, hormones are chemical messengers produced and secreted by specialized cells of the body called glands. These chemical messengers send out messages to influence or effect cells in other parts of the same organism. All multicellular organisms produce hormones. Plant hormones are referred to as phytohormones. In animals, hormones are often transported in the blood to cells through specific receptors to respective cells or organs. To carry out its functions a hormone binds to its specific protein receptor to induce the activation of a signal transduction mechanism that alternately leads to cell type specific responses. Signal transduction is a mechanism that converts a mechanical or chemical stimulus to a cell into a specific cellular response (Reece and Campbell, 2002). It starts with a signal to a receptor and ends with a change in cell function. Transmembrane receptors are outside and some inside the cell. The chemical signals (hormones) bind to the portion of the receptor, changing its shape and conveying another signal inside the cell. Some chemical messengers such as testosterone can pass through the membrane and bind directly to receptors in the cytoplasm or nucleus. Some times, there is a cascade of signals within the cell. With each of these cascades, the signal can be amplified, so a small signal can result in a large response (Reece and Campbell, 2002). Eventually the signal creates a change in the cell, either in the expression of the DNA in the nucleus or in the activity of enzymes in the cytoplasm. These processes can take milliseconds for ion flux and minutes for protein and lipid mediated cascades by producing a “second messenger” molecule signal into the signal biochemical network.

The “second messengers” such as cAMP.cGMP, Ca 2+ diacylglycerol (DG), activate respective protein kinase of the living organism (Reece and Campbell, 2002). Protein kinases are divided into serine/threonine and tyrosine specific kinases (Beato et al., 1996). While the kinases catalyse the phosphorylation of the receptor proteins, the phosphatases catalyse the dephosphorylation. The phosphorylation and dephosphorylation cycle enables the cells to alternate from the resting to activated state and vice versa, according to the stimuli impute. Protein phosphorylation changes enzyme activities and protein conformation. The eventual outcome is alteration in cellular activities and changes in the programme of genes expression within the responding cells. Through this signal transduction biochemical processes, the hormones affect or influence changes in the cells and organs of the body. In human, there are the endocrine and exocrine hormones. For the purpose of this work, our interest is more on the endocrine hormones.

1.5.1 Follicle Stimulating Hormone

The FSH is a hormone found in animals and human. It is synthesized and secreted by gonadotrophs of the anterior pituitary gland. FSH regulates the development, growth, pubertal maturation and reproductive processes of the body. FSH and Luteinizing hormone (LH) act synergistically in reproduction.FSH is a glycoprotein. Each monomeric unit is a protein molecule with carbohydrate molecule (sugar) attached to it. Two of these make a full functional protein. The protein dimer contains two polypeptide units, labeled alpha and beta subunits. The alpha subunits of FSH contain 92 amino acids. The beta subunits vary. FSH has beta subunits of 118 amino acids which confers its specific biologic action and is responsible for interaction with FSH receptor. The sugar part of the hormone is composed of fructose, galactose, galactosamine, mannose, glucosamine and sialic acid. Sialic acid is critical for its biologic half-life. FSH half-life is 3-4hrs and its molecular weight is 3000. The genes for alpha subunits is located on chromosome 6p21.1-23. It is expressed in gonadotropes of pituitary cells, controlled by gonadotropin releasing hormone, inhibited by inhibin and enhanced by activity. Inhibin is a non steroidal polar substance of testicular origin that prevents hypertrophy of the hypophysis in male animals while activity is a protein complex produced in the gonads, pituitary gland and placenta. They alternate the biosynthesis and secretion of FSH. The FSH regulates the development, growth, pubertal maturation, and a reproductive process of the human body. FSH exerts these actions by binding to specific receptors, localized exclusively in the gonads. The FSH receptors belong to the family of G-protein coupled receptors. They are complex membrane proteins characterized by seven hydrophobic helices inserted in the plasmalemma and by intracellular and extracellular domains of variable dimensions depending on the type of ligands. The intracellular portion of the FSH receptors is coupled to a G protein and, upon receptors activation by the hormonal interaction with extracellular domain to the specific biological effects of the gonadotropin (Chappel and Howles, 1991; Moyle and Campbell, 1995; Ulloa-Agure et al., 1995). In both males and females, FSH stimulates the maturation of germ cells. In males, FSH induces the sertoli cells to secret inhibin and stimulates the formation of sertoli tight junctions referred to as Zonula occludens. In females, FSH initiates follicular growth, specifically affecting granulosa cells. With the concomitant rise in inhibin B, FSH levels then decline in the late follicular phase. This seems to be critical in selecting only the most advanced follicle to proceed to ovulation. At the end of the luteal phase, there is a slight rise in FSH that seems to be of importance to start the next ovulatory cycle. The FSH release at the pituitary gland is controlled by pulses of the Gonadotropin releasing hormone (GnRH). Those pulses are in turn subjects to oestrogen feedback from the gonad. Apart from stimulating the growth and recruitment of immature ovarian follicles in the ovary, FSH is the major survival factor that rescues the early (small) antral follicle from apoptosis. In the luteal-follicular phase transition period, the serum level of progesterone and oestrogen (primarily oestradiol) decrease and no longer suppress the release of FSH. Consequently, FSH peaks at about day three of the menstrual flow. The cohort of small antral follicles is normally sufficiently in number to produce enough inhibin B-to lower the FSH serum levels. As a woman nears perimenopause, the number of small antral follicles recruited in each cycle diminishes and consequently, insufficient inhibin B is produced to fully lower FSH and the serum level of FSH begins to rise. When the follicle matures and reaches 8-10mm in diameter, it begins to secret significant amounts of oestradiol. In humans only one follicle becomes dominant and survives to 18-30mm in size and ovulates while the remaining follicles in the cohort undergo atresia. The sharp increase in oestradiol production by the dominant follicle cause a positive effect on the hypothalamus and pituitary and rapid GnRH pulses occur and a LH surge results. The increases in oestradiol levels cause a decrease in FSH production by inhibiting GnRH production in the hypothalamus. The decrease in serum FSH level causes the smaller follicles in the cohort to undergo atresia as they lack sufficient sensitivity to FSH to survive. Occasionally two follicles reach 10mm stags at the same time by chance and both are equally sensitive to FSH. Both survive and grow in low FSH environment and thus two ovulations can occur in one cycle, possibly leading to non-identical (dizygotic) twins. In males, FSH stimulates maturation of seminiferous tubules and spermatogenesis. FSH enhances the production of androgen-binding protein by the sertoli cells of the testes by binding to FSH receptors on their basolateral membrane (Walter, 2003) and is critical for initiation of spermatogenesis. FSH measurement should be typically and optimally measured on day three of a woman’s cycle when the levels of oestradiol and progesterone are at lowest point of the menstrual cycle.

The most common reason for high serum FSH concentration is a female who is undergoing or who has recently undergone menopause. High level of FSH indicate that the normal restricting feed back from the gonad is absent, leading to an unrestricted pituitary FSH production. If high FSH level occurs during the reproductive years, it is abnormal and this can be seen in the following conditions, premature menopause also known as pre mature ovarian failure, poor ovarian reserve referred to as premature ovarian ageing, Gonadal dysgenesis, turners syndrome, castration in male, Swyers syndrome, testicular failure and certain forms of congenital adrenal hyperplasia (CAH). Most of conditions are associated with subfertility and/or infertility. Diminished secretions of FSH can result in failure of the gonadal functions (Hypogonadism). This condition is typically manifested in males as failure in production of normal number of sperm. In females, cessation of reproductive cycles is commonly observed. Conditions with very low FSH secretion are: polycystic ovarian syndrome in which there are many small tumours in the ovary, presenting as benign or malignant cystic tumors, obesity, hirsutism and infertility, kallmann syndrome or hypothalamic hypogonadism (decreased function of the glands that produce sex hormones), hypothalamic suppression, hypopituitarism, hyperprolactinemia and gonadotropin deficiency. Also in gonadal suppression therapy where GnRH antagonists or agonists (for down regulation) are given, FSH level is suppressed. There is a significant association between FSH levels and sleep duration. Follicle stimulating hormone levels were 20% higher in long-time sleepers than in short-time sleepers. This association persisted whatever the age or the body mass index. This therefore implies that the longer the sleep of an individual is deprived, the lower the level of FSH. During five days of military traning course for male cadets hard physical activity in the day and night sleep deprivation. It was discovered that LH, FSH and prolactin and TSH wave decreased (Opstad, 1992). In males, prolonged sleep deprivation subsequently decreases the availability of testosterone to target organs, thereby creating a condition of hypospermatogenesis, oligospermia and male infertility. In females also the prolonged sleep deprivation lowers the FSH levels with consequent deficient follicular development, anovulation and infertility (Tauzet et al., 2002).

1.5.2 Luteinizing Hormones

Luteinizing hormone (LH), also known as Lutropin is a hormone produced by the adenohypophysis of anterior pituitary gland. LH is a heterodimeric glycoprotein. Each monomeric unit is a glycoprotein molecule. One alpha and one beta subunit make the full functional protein just as in FSH. The protein dimer contains two glycopeptide sub units, labeled alpha and beta subunits that are non-covalently associated. It also contains 92 amino acids in human but 96 amino acids in almost all other vertebrates.The beta subunit of LH has 121 amino acids that confers its specific biological action and is responsible for the specificity of the interaction with LH receptor. The beta subunit contains an amino acid sequence that exhibit large homologies with that of beta subunit of human chorionic gonadotropin (HCG) and both stimulate the same receptor. However, the HCG beta subunit contains an additional 24 amino acids, and the two hormones differ in composition of their sugar moieties. The different composition of these oligosaccharides affects bioactivity and speed of degradation of this hormone. The biologic half-life of LH is 20 minutes. The gene for alpha sub units is located on chromosome 6q 12:21 while the beta subunits gene is localized in chromosome 19q 13:32. The molecular weight of LH is 28,500. In contrast to the alpha subunit gene activity, beta LH sub unit gene activity is restricted to the pituitary gonadotropic cells. It is regulated by the gonadotropin releasing hormone from the hypothalamus. Inhibin, activin and sex hormones do not affect genetic activity for the beta subunit production of LH. Mechanism of action of luteinizing hormones on target cells involves the roles of gonadotropin receptors, adenosine monophosphate (AMP), prostaglandin protein kinas, cyclic Adenosine monophosphate (cAMP) and cyclic Adenosine 3’,5’-monophosphate (Channing and Tsafriri, 1977). The luteinizing hormone acts by binding to specific receptors in the target cells, the ovaries and the testes. In the testes the receptor complexes have apparent molecular weight of 200,000 Kda. The binding of all gonadotropin hormones to cells generally increase the activity of plasma membrane-bound adenylate cyclase.In addition to the LH stimulatory effect on the adenylate cyclase of corpora lutea and leydig cells, it increases the synthesis of one of the prostaglandins of the E group, which can decrease or increase the synthesis cAMP (White et al., 1978). It should be noted that the mechanism by which luteinizing hormone stimulates the synthesis of ovarian steroid hormones is a subject of controversy. Mckerns has proposed that a primary action of this gonadotropin in rats’ lutein and bovine corpus luteum cells is to raise the rate of production of reduced nicotinamide-adeninedinucleotide phosphate (NADPH) by a direct stimulation of glucose-6-phosphate dehydrogenase. Flunt and Denton (1970) have concluded that luteinizing hormone stimulates steroid production by an effect on protein synthesis following an increase in the intracellular concentration of cyclic 3’, 5’-Ad enosine monophosphate.

In both male and female, LH is essential for reproduction. In females, at the time of menstruation, FSH initiates follicular growth specifically affecting granulosa cells. With the rise in oestrogens in this process, LH receptors are also expressed on the maturing follicle that produces an increasing amount of oestradiol. Eventually, at the time of the maturation of the follicle, the oestrogen rise leads to the positive feed back effect via the hypothalamus and a release of LH over a 24-48 hour period. This “LH surge”triggers ovulation thereby not only releasing, but also initiating the conversion of residual follicle into a corpus luteum that in turn produces progesterone to prepare endometrium for implantation. LH is necessary for maintenance of luteal function for the first two weeks. In case of a pregnancy, luteal function will be further maintained by the action of HCG (a hormone very similar to LH) from the newly established pregnancy. LH supports thecal cells in the ovary that provide androgens and hormonal precursors for oestradiol production. In males, LH acts on leydig cells of the testes to produce testosterone. Testosterone is an androgen that exerts both endocrine activity and intratesticular activity on spermatogenesis. The release of LH at the pituitary gland is controlled by pulses of gonadotropin-releasing hormone (GnRH) from the hypothalamus. Those pulses are in turn subject to the oestrogen feedback from the gonads. LH levels are normally low during childhood and, high after menopause in women. As LH is secreted as pulses, it is necessary to follow its concentration over a sufficient period of time to get a proper information about its blood level. During the reproductive years, typical levels are between 1-20 IU/L. Physiologic high levels are seen during the LH surge which last 48 hours. The detection of the luteinizing hormone surge indicates impending ovulation and that is the principle used by urinary ovulation predictor kit or LH kit. This test is performed daily around the time ovulation is expected. The conversion from a negative to positive result would suggest that ovulation is about to occur within 24-48 hours, giving women, two days to engage in coitus or artificial insemination with intention of conceiving (Neilsen et al., 2001).

A number of disease conditions are associated with abnormal levels of LH in both male and females. Precocious puberty is an abnormal condition where children develop reproductive features much earlier than they suppose because their LH and FSH levels got to reproductive range instead of the low levels typical of their age. This in some cases may be as a result of tumor or injury of the brain. This unusual development may often affect social behavior, psychological development, may reduce adult height potentials and shift some life long health risks. This could be of pituitary or central origin. The central precocious puberty is usually treated by suppressing the pituitary hormones that induces the sex steroids. During the reproductive years, relatively elevated levels of LH is seen in patients with the polycystic ovarian syndrome and persistently high levels of luteinizing hormone are indicative of absence of the normal restricting feedback from the gonads. While this condition is normal in menopausal life, it is very abnormal in reproductive years, and may be indicative of the following infertility conditions: premature menopause, Gonadal dysgenesis Turner’s Syndrome, castration, Swyer Syndrome polycystic ovarian Syndrome, certain forms of CAH and testicular failure. Persistently low level of LH on the other hand may present fertility problems such as kallmann Syndrome, Hypothalamic suppression, Hypopituitarism, Eating disorder, female athlete triad (disordered eating, amenorrhea and osteoporosis seen among females athletes), Hyperprolactinemia, gonadotropin deficiency to mention but a few (Kaplan and Pasce, 1989). Generally infertility can be induced in male and females by sleep deprivation. Stress arising from from sleep deprivation can disrupt the hormonal communication between the brain, the pituitary, and the ovary or the testes, thereby interfering with maturation of eggs, ovulation, and spermatogenesis and sperm secretion as the case may be. In a work done by Strassman et al. (1991) on 17 normal men, it was reported that sleep deprivation reduced secretion of LH. It was also reported that sleep apnoea and narcoleptic men have reduced LH secretion. However, it is important to note that some researchers reported that in the study of sleep deprivation on the pituitary-testis axis physiology of eight healthy men, there was no decrease in FSH, LH and prolactin but there was significant decrease in testosterone and Oestradiol. In another research on the influence of partial sleep deprivation on the secretion of thyrotropin, thyroid hormones, growth hormone, prolactin, LH, FSH and oestradiol in healthy young women, there was decrease in prolactin, increase in TSH, LH and E2 concentrations while FSH and GH remained unchanged (Baumgartner et al., 1993). During a five days military training course for male cadets with hard physical activity day and night and almost no sleep and food, a decrease was found in LH, FSH, PRL and TSH (Opstad, 1992).

1.5.3 Prolactin (PRL)

Prolactin or luteotropic hormone (LTH) is a peptide hormone discovered by Dr Henry Friesen. It is primarily associated with lactation (Mancini et al., 2008). In breast feeding, the act of an infant sucking the nipple stimulates the production of prolactin, which fills the breast, with milk via a process known as lactogenesis, in preparation for the next feed. Oxytocin is another hormone which triggers milk let-down. Prolactin is synthesized and secreted by lactotroph cells in the adenohypophysis (anterior pituitary gland). It is also produced in other tissues including the breast and deciduas.

Prolactin is a single chain polypeptide of 198 amino acids with a molecular weight of about 24,000 kilodaltons. Its structure is similar to that of growth hormone and placental lactogen. The molecule is folded due to the activity of three disulfide bonds. The molecule has significant heterogeneity in glycosylation, phosphorylation and sulfation and as a result, the bio assays and immunoassays of prolactin can give different results. The non-glycosylated form of prolactin is the dominant form of prolactin that is secreted by the pituitary gland. The prolactin binds to prolactin receptors for its effects to be carried out on the target cells. Prolactin receptors are present in mammary glands, ovaries, pituitary glands, heart, lungs, thymus, spleen, liver, pancreas, kidney, adrenal gland, uterus, skeletal muscle, skin and areas of the central nervous system (Mancini et al., 2008). When prolactin binds to the receptor it causes it to dimerize with another prolactin receptor. This result in the activation of Janus Kinase 2, a tyrosine Kinase which initiates the Jak-Stat pathway. The activation of prolactin receptor also results in the activation of mitogen-activated protein kinase and src kinase (Mancini et al., 2008). The pituitary prolactin secretion is regulated by neuroendocrine neurons in the hypothalamus, most importantly by neurosecretory dopamine neurons (tuberoinfundibular) of the arcuate nucleus, which inhibit prolactin secretion. Dopamine secreted here acts on the dopamine-2-receptor of the lactotrophs, causing the inhibition of prolactin secretion. Thyrotropin releasing factors (thyrotropin releasing hormone) and serotonin have stimulatory effects on prolactin release. Also the vasoactive intestinal peptide and peptide histidine isoleucine help to regulate prolactin secretion in humans, but the function of these hormones in birds can be quite different (Kulick et al., 2005). Prolactin has many effects including lactation, orgasm, and stimulating proliferation of oligodendrocyte precursor cells. Prolactin also stimulates proliferation of corpora lutea and stimulation of progesterone secretion. Prolactin actually acts synergistically with estrogen to promote the mammary gland proliferation. An increased serum concentration of prolactin during pregnancy causes the enlargement of the mammary glands of the breast and increases the production of milk. As a result of increased level of prolactin, there is also a high level of progesterone during pregnancy and these acts directly on the breasts to stop ejection of milk. It is only when the levels of this hormones fall after child birth that milk ejection is possible. Sometimes new born babies (Males and females) secret a milk substance from their nipples. This substance is commonly known as witch’s milk. This is caused by the fetus being affected by prolactin circulation in the mother: first before birth and usually stops after birth. Prolactin provides the body with sexual gratification after sexual acts (Obal et al., 1997; Zhang et al., 2001). The hormone counteracts the effect of dopamine, which is responsible for sexual arousal. This is thought to cause the sexual refractory period. The amount of prolactin can be an indicator for amount of sexual satisfaction and relaxation. Usually high amount is suspected to be responsible for impotence and loss of libido as in hyper prolactinamiae symptoms (Gregg et al., 2007).

Prolactin exhibits an antigonadotropic hormone action such as inhibition of luteinization by luteinizing hormone and the ovulation induced by pregnant mare’s serum. The antiovulatory action of prolactin depends on the presence of corpora lutea, suggesting that the effect on ovulation is secondary to an influence on progesterone secretion by the corpus luteum (White et al., 1978). Prolactin has a number of other effects including contributing to surfactant synthesis of fetal lungs at the end of pregnancy and immune tolerance of the fetus by maternal organism during pregnancy. It also decreases normal levels of sex hormones; estrogen in women and testosterone in men (Kaplan and Pesce, 1989). The inhibition of sex steroids is responsible for loss of menstrual cycle in lactating women as well as lactation-associated osteoporosis. Prolactin also enhances luteinizing hormone receptors in the leydig cells, resulting to testosterone secretion, which leads to spermatogenesis. Apart from the reproductive system, prolactin stimulates the proliferation of oligodendrocytes. These are nervous tissue cells that generate neurons and astrocytes, which support axons and produce myelin sheath. Myelin sheath insulates and lowers the effective capacitance of axons in the CNS (Gregg et al., 2007). Variance in levels exist in the blood circulation of prolactin. There is a diurnal as well as ovulatory cycle in prolactin secretion. There is also a seasonal change in prolactin release. During pregnancy, high circulatory concentrations of estrogen promote prolactin production. The resulting high level of prolactin secretion causes further maturation of the mammary glands, preparing them for lactation. After child birth, prolactin levels fall as the internal stimulus for them is removed. Sucking by the baby on the nipple then promotes further prolactin release, maintaining the ability to lactate. The sucking activates the mechano receptors in and around the nipple. These signals are carried by the nerves fibers through the spinal cord to the hypothalamus, where changes in the electrical activity of neurons that regulate pituitary gland cause increased prolactin secretion. The sucking stimulation also triggers the release of oxytocin from the posterior pituitary gland, which induces milk let down. Prolactin controls lactogenesis (milk production) but not milk ejection reflex. The rise in prolactin fills the breast with milk in preparation for the next feed. In usual circumstances, in the absence of galactorrhea, lactation will cease within one or two weeks of the end of demand for breastfeeding. It has also been found that compared to unmated males, fathers and expectant fathers have increased prolactin concentrations (Nelson, 2005). Following the WHO standard, reference ranges for prolactin is 2.8-29.2ng/ml in females and 2.1-17.7ng/ml in males. Prolactin levels peak during rapid eye movement (REM) sleep, and in the early morning. Levels can rise after exercise, meals, sexual intercourse, minor surgical procedures and following epileptic seizures (Melned and Jameson, 2005; Mellors, 2005). It is noteworthy that high prolactin levels tend to suppress the ovulatory cycle by inhibiting FSH and GnRH and can contribute to mental health issues. A condition associated with elevated prolactin in the blood is termed hyperprolactinemia. This can result from prolactinoma, a highly elevated level of prolactin found in some pituitary tumors, resulting to amenorrhea and infertility. Others include excess thyrotropin-releasing hormone (TRH), usually found in hypothyroidism, emotional stress, antipsychotic medications, pregnancy and lactation and in some sexual disorders.

Conditions associated with decreased prolactin level in the blood are seen in bulimia, an eating disorder involving repeated episode of uncontrolled consumption of large quantities of food in a short time and in excess dopamine. Sleep and sleep deprivation showed some variations of prolactin levels thus: the secretion of prolactin and growth hormone was investigated during sleep in 10 healthy volunteers and this showed that prolactin secretion is entrained into sleep cycle of Non-REM and REM periods. A maximum of plasma hormone elevations occurs during the first quarter of sleep cycles i.e. during Non-REM periods and less frequent rises at the end of the cycles, mainly during REM periods in contrast to growth hormone concentration of prolactin remain higher also during later cycles occurring towards morning. This shows that high prolactin regularly occur during sleep cycles with small amount of slow wave sleep. Maximal prolactin concentrations during sleep are not affected by selective sleep deprivation of slow wave sleep (SWS) stages 3 and 4. This is further evidence that slow wave sleep stages are not necessary for the development of high plasma prolactin concentration (Beck et al., 1976). In an experiment of partial sleep deprivation, increases were observed in the levels of prolactin, Luteinizing hormones, and oestradiol (Baumgartner et al, 1993). However, in another investigation of hypothalamo-pituitary regulation of androgen secretion in young men after prolonged physical stress and sleep deprivation for five days, a decrease was found in LH, FSH, Prolactin and TSH. A decrease was also found in testosterone, dihydrotestosterone (DHT), androstenedione, dehydroepiandrosterone and 17 α-OH progesterone (Opstad, 1992). The influence of partial sleep deprivation on the secretion of TSH, T4, FT4, T3, prolactin, GH, LH, FSH and oestradiol was investigated in 10 healthy young women. Blood samples were drawn at hourly intervals over a 64 hour period i.e. 3 consecutive days at night. Among other effects on other hormones, prolactin levels decreased (Baumgartner et al, 1993). In another experiment during prolonged physical training and sleep deprivation on eleven young male cadets participating in a 5 days ranger training course, prolactin testosterone and oestradiol strongly decreased (Opstad and Aakvaag, 1982). In a research work done by Carol A. Everson and colleague on circulatory anabolic hormones, the result indicated that prolactin, GH, insulin- like growth factors (IGF) and leptin were suppressed by 15 day sleep deprivation (Partial and total sleep deprivation) in rats (Everson and Crowley, 2004).

1.5.4 Thyroid Stimulating Hormone

(The thyroid stimulating hormone (TSH), also known as thyrotropin is a peptide hormone synthesized and secreted by thyrotrope cells in the anterior pituitary gland, which regulates the endocrine function of the thyroid gland (Sarcher and McPherson, 2000). TSH is a glycoprotein and consists of two non covalently bound subunits, the alpha (α) and the beta (β) subunits. The alpha subunit (Chorionic gonadotropin alpha) is identical to that of human chorionic gonadotropin (HCG), luteinizing hormone and follicle stimulating hormone. The beta subunits are unique to TSH and therefore determine its function. TSH initiates its action through the TSH receptor. TSH has a molecular weight of 28 300 Kda. The α-subunit has a molecular weight of 13600 Kda while that of β sub units is 14,700 Kda. This hormone is among the proteins richest in sulfur content, having 11 disulfide linkages but no free sulfhydryl groups. The latter are all intrachain bridges, 5 in the α and 6 in B (White et al., 1978).

The biological activity and influence of TSH or the target cell is initiated and achieved by the binding of the TSH on the TSH receptors (TSHR). The TSHR is found mainly on the thyroid follicular cells (Parmentier et al., 1989). The TSHR gene, cloned in 1989, maps to human chromosome 14q and encodes a predicted seven transmembrane, G protein-coupled glycoprotein (Parmentier et al., 1989; Nagayama et al., 1989). Although, it is similar to LH and FSH receptors, the TSHR is the largest of the glycoprotein hormone receptors due primarily to 8 and 50 amino acid insertions in the ectodomain (residue 38-45 and 17-367) (Rapoport et al., 1998).

The TSHR bound to TSH signals action via cAMP (9 of the TSH receptor) and stimulates both the phospholipase C (PLC) and protein kinase signal transduction systems. Intracellular Ca2+ and PLC regulate iodide efflux, H2O2 production and thyroglobulin iodination while adenylate cyclase and cAMP regulate iodide uptake and transcription of thyroglobulin (TG), thyroid peroxidase (TPO) and the sodium-iodide symporter (Field et al., 1987; Riedal et al., 2001). These actions lead to the production of T3 and T4 in the thyroid gland. TSHR levels and actions are regulated by negative feedback mechanism of T3 and T4 concentrations in the systems. Apart from the regulation of thyroid hormones (T3 and T4), and its role in controlling the growth and development of the thyroid gland, TSHRs have been discovered to exhibit wider functional roles. This is because of its identification in a number of other tissues such as the brain, testes, kidney, heart, bone, thymus, lymphocytes, adipose tissues and fibroblasts (Davies et al., 1978). This secretion has a direct proportionality influence on thyroid hormone production to a peak high level beyond which the thyroid hormones exert a negative feed back effect on TSH production through the TRH in the hypothalamus. The total morphological changes resulting to infertility caused by hypothyroidism is being traced to a consequence of higher prolactin production that can block the secretion and action of gonadotropin, being the main cause of the changes observed (Amada-Dias et al., 2001). In women, hypothyroidism is associated with delay in the onset of puberty, anovulation, amenorrhoea, menstrual irregularity, infertility, increased frequency of spontaneous abortions (Amada-Dias et al., 2001). It was suggested that these alterations may be caused by a decrease in LH secretion, consequent on hyperprolactinemia. This decreases GnRH secretion, LH frequency and pulsatility; counteracts the morphological effects of LH in culture of granulosa cells, having a luteolytic effect and causing inhibition of folliculogenesis, estrogen synthesis and ovulation (Amada-Dias et al, 2001). This could explain the decrease in gonadotropin stimulation in the ovaries of hypothyroid women. The fact that the ovarian follicles were not well developed in the hypothyroid animals despite normal serum LH, FSH, oestradiol and progesterone suggests that thyroid hormones could have a direct effect on the growth of ovarian follicles, without a significant effect on the sex steroid production by the ovaries.Infact, T3 receptors were found in the granulosa cells of Porcine and human ovaries. Thyroid hormones increase the action of FSH in culture of porcine granulosa cells, suggesting a direct effect of T3 on the ovaries (Amadas-Dias et al., 2001).

Just like the hormones already discussed sleep deprivation has a physiological effects on TSH. During a five days training course for male cadet officers with hard physical activity and almost no sleep, TSH levels among others decreased (Opstad, 1992). In an investigation of the alterations of pituitary dependent hormones during sleep loss, it was stated that after 6 days of sleep restriction (4 Hours of bedtime) and after full sleep recovery (6 days of 12 hours bed time), the normal nocturnal thyroid stimulating hormones levels rise was strikingly decreased and the overall mean TSH levels were reduced by more than 30%. A normal pattern of TSH release reappeared when the subjects had fully recovered. Differences in the TSH profiles between the 2 bed time conditions were probably related to changes in thyroid hormone concentration via a negative-feedback regulation, because the free thyroxine index (FT41) was higher in sleep restriction condition than in fully rested condition (Vancauter et al, 2009). Also according to Van Cauter and Colleagues in the science daily of 1999, sleep deprivation dampened the secretion of TSH and increased the blood levels of cortisol especially during afternoon and evening (Vancauter, 1999). On the contrary in a study of total sleep deprivation and the thyroid axis, it was noted that sleep deprivation was associated with elevated TSH (Gary et al, 1996). Also in research carried out by Andreas Baumgartner et al, 1993, on the influence of partial sleep deprivation on the secretion of thyrotropin (TSH), thyroid hormone, FSH and oestradiol on 10 healthy young women, it was observed that TSH concentrations increased significantly and remained elevated throughout the following day but levels in T3, T4 and FT4 were enhanced during the partial sleep deprivation only and changes in these hormones seemed to be independent of TSH. In another research on nocturnal TSH and prolactin secretion during sleep deprivation and prediction of antidepressant response in patients with major depression, TSH levels increased significantly during total sleep deprivation while prolactin levels decreased significantly. This agrees with the knowledge of the physiology of sleep which states that in normal adult men and women, TSH levels are low through the day time and begins to increase in the late afternoon or early evening.Maximal levels occur shortly before sleep. During sleep, TSH levels generally decline slowly. A further decrease occurs in the morning hours, studies involving sleep deprivation and shifts of sleep wake cycle have consistently indicated that an inhibiting influence is exerted on TSH secretion during sleep. Interestingly when sleep occurs during the daytime hours, TSH secretion is not suppressed significantly below normal day time levels. Sleep deprivation at night periods increases TSH levels. Also under conditions of sleep deprivation, there is increased amplitude of TSH rhythm resulting in increased amplitude of Triiodothyronine, T3 rhythm (Parker et al, 1987). Following the observation above, on TSH, the actual effect of sleep deprivation TSH is still inconclusive.

1.5.5 Testosterone

Testosterone is a steroid hormone from the androgen group of hormones. It is found in mammals, reptiles, birds and other vertebrates (Cox and John-Alder, 2005; Reed et al, 2006). In mammals, testosterone is primarily secreted in the testes of males and ovaries of females. Small amount is also secreted by the adrenal glands. Testosterone is the principal male sex hormone and an anabolic steroid. In men, testosterone plays a key role in the development of male reproductive tissues such as testis, penis, the scrotum, prostate, and seminal vesicles. It facilitates the development of secondary sex characteristics such as musculature and bone mass, hair growth and patterns, fat distribution, laryngeal enlargement and vocal cord thickening. Additionally, normal testosterone levels maintain energy level, healthy mood, fertility and sexual desires (Basil et al, 2009) as well as prevention of osteoporosis (Tuck and Francis, 2009). Testosterone is conserved through most vertebrates although fish make a slightly different form called 11-ketotestosterone. Its counterparts in insects are ecdysone (Arnold, 2006). Testosterone has wide physiological effect in both male and females. In general, androgens promote protein synthesis and growth of these tissues with androgen receptors. Testosterone effects can be classified as virilizing (Having the strength that is considered typical of men and also sexual energy) and anabolic, meaning that it builds up bone and muscle mass. The growth muscle mass and strength, increased bone density and strength, and stimulation of linear growth and bone maturation are the known anabolic effects. The androgenic effect includes the maturation of the sex organs, particularly, the penis and the formation of scrotum in the fetus. After birth, usually at puberty, it causes a deepening of voice, growth of the beard and axillary hair. Most of the prenatal androgen effects occur between 7 and 12 weeks of gestation, and they include genital virilization (Midline fusion, phallic urethra, and scrotal sac thinning and rugation phallic enlargement) development of prostate and seminal vesicles and gender identity. However, the role of testosterone in virilization is far smaller than that of dihydrotestosterone. Early infancy androgenic effects are the least understood. In the first weeks of life for male infants, testosterone begins to rise. The levels remain in pubertal range for detectable levels of child hood by 4 to 6 months of age (Forest et al., 1973).

It has been speculated that brain masculinization begins to occur at this stage since no significant changes have been identified in other parts of the body. Surprisingly, the male brain is masculinized by testosterone being aromatized into estrogen which crosses the blood brain barrier and enters the male brain where as female fetuses have alpha-fetoprotein which binds up the estrogen so that female brain are not affected (Utexas, 1998). The pre-pubertal effects of testosterone are the observable effects of rising androgen levels at the end of child hood. This occurs in both boys and girls with observable signs such as adult type body odor, increased oiliness of skin, hair and acne, puberch (appearance of pubic hairs), auxiliary hair, growth spurt, accelerated bone maturation and hair on upper lip and side burns.

Pubertal effects begin to occur when androgen has been higher than normal adult female levels for months or years. In males, these are usually late pubertal effects, and occur in women after prolonged periods of heightened levels of free testosterone in the blood. These pubertal effects manifest as enlargement of sebaceous glands which might cause acne, phallic enlargement or clitolomegaly, increased libido and frequency of erection or clitoral engorgement, extention of public hairs to thighs and up towards umbilicus, facial hairs (side burns, beard, moustache), loss of scalp hair (Adrogenic alopecia), chest hairs, periareolar hair, perianal hair, leg hair, auxiliary hair, decrease of facial subcutaneous fat increased muscle strength and mass,deepening of voice, Adam’s apple growth of spermatogenic tissues in the testicles, growth of jaw, brow, chin, nose, remodeling of facial bone contours, broadening of shoulders, expansion of rib cage, completion of bone maturation and termination of growth. These occur via oestradiol metabolites and hence more gradually in men than in women.

In adults, testosterone effects are more clearly demonstrated in males than in females but are important to both sexes. Some of these effects may decline as testosterone levels decrease in the later decades of adult life. The effects include normal libido and clitoral engorgement/penile erection frequency, regulation of acute HPA response under dominance challenge (Mehta et al., 2008), mental and physical energy, maintenance of muscle trophism. Normal testosterone maintenance in elderly men has been shown to improve many parameters which are thought to reduce cardiovascular disease risk, such as increased lean body mass, decreased visceral fat mass, decreased total cholesterol and glycermic control (Haddad et al., 2007).

Under dominance challenge, testosterone may play a role in the regulation of fight-or-flight response. It regulates the population of thromboxane A2 receptors on megakaryocytes and platelet aggregation in humans. Testosterone is necessary for normal sperm development. It activates genes in the sertoli cells, which promote differentiation of spermatogonia. Studies show that falling in love decreases men’s testosterone levels while increasing women’s testosterone levels. It is speculated that these changes in testosterone results in the temporary reduction of differences in behavior between the sexes (Sapienza et al., 2009).

Recent studies suggest that a testosterone level plays a major role in risk taking during financial decisions (Sapienza et al., 2009). Fatherhood is said to decrease testosterone levels in men, suggesting that the resulting emotional and behavioral changes promote paternal care. Testosterone affects the entire body, often by enlargement. As a result, men have bigger hearts, lungs, liver etc, and even larger brain, possibly because of higher testosterone levels (Cosgrove et al., 2007). The enzyme aromatase converts testosterone into oestradiol that is responsible for masculinization of brain in male mice. In a Danish study from 2003, men were found to have a total myelinated fiber length 176,000km at age of 20, where as in women, the total length was 149,000km (Marner et al., 2003). However, women have more dendritic connections between brain cells. Literature suggests that attention, memory and spatial ability are key cognitive functions affected by testosterone in humans. Preliminary evidence suggests that low testosterone levels may be a rik factor for cognitive decline and possibly for dementia of the Alzheimer’s type (Moffat et al., 2004). Testosterone depletion is a normal consequence of aging in men and one possible consequence of this as mentioned earlier could be an increased risk for the development of Alzheimer’s disease.

The biochemistry of production and action of testosterone is well elucidated. Like other steroid hormones, testosterone is derived from cholesterol. The first step in the biosynthesis of testosterone involves the cleavage of the side chain of cholesterol by Cyp11A, a mitochondrial cytochrome P450 oxidase with the loss of six carbon atoms to give pregnenolone. In the next step, two additional carbon atoms are removed by the CYP17A enzyme in the endoplasmic reticulum to yield a variety of C19 steroids (Zuber et al., 1986). In addition, the 3-hydroxyl group is oxidized by 3-β-HSD to produce androstenedione. In the final and rate limiting step the C17 keto group androstenedione is reduced to yield testosterone.

The largest amount of testosterone (>95%) are produced by the testes in men (Mooradian et al., 1987), while a far smaller amount is synthesized in women by the thecal cells of the ovaries and by the placenta. It is also produced by the zona reticularis of the adrenal cortex in both sexes. In the testes, testosterone is produced by the Leydig cells (Brooks, 1975). The male generative glands also contain sertoli cells which require testosterone for spermatogenesis. Approximately 7% of testosterone is reduced to 5 α-dihydrotestosterone (DHT) by the cytochrome P450 enzyme, 5 α—reductase an enzyme highly expressed in male accessory sex organs and hair follicles (Mooradian et al., 1987,). Approximately 0.3% of testosterone is converted into oestradiol by aromatase (Cyp19A) (Meinhardt and Mullis, 2002), an enzyme expressed in the brain, liver and adipose tissues (Mooradian, 1987). DHT is a more potent form of testosterone. While testosterone has the activities of masculinization, oestradiol has feminization roles. However an appropriate amount of estrogen is required in the male in order to ensure well being, bone density, libido, erectile function,to mention but a few.

Since it has been reported that systemic disorders resulting from prolonged sleep deprivation include certain changes in pituitary hypothalamus axis and hormone secretion (Bergmann et al., 1989), its effects on sex hormones such as testosterone and oestradiol are in this context worthy of note. Testosterone levels of healthy men decline as they get older. As sleep and sleep quality and quantity typically decrease with age, Plamen Penev conducted a study in which he objectively measured differences in the amount of sleep. The result of this study raised the possibility that older men who obtain less actual sleep during the night have lower blood testosterone levels in the morning (Penev, 2007). In a another study on the effect of sleep deprivation and high calorie diet on eleven young male cadets, participating in a 5 days rangers training course involving heavy and continuous physical activities and sleep deprivation , testosterone was found to be decreased during the day time and reached below 25% of precourse value after 48 hrs (Opstad and Aakvaag, 1982). A diversity of aversive stimuli applied to animal models on a short-or-long term basis have been used to investigate the response of HPA and/or gonadal axis to stress (Almeida et al., 2000). Stress related hormones are known to modulate male reproductive functions by interfering with the hypothalamic pituitary-gonadal axis. Consequently, secretion of sex steroids is altered (Almeida et al., 2000; Knol, 1991). Also a significant decrease in both the production of maturing spermatids and testicular maturation has been described in rats submitted to immobilization stress (Almeida et al., 2000). Stress impairs gonadal function and lowers testosterone levels. Moreover, reduced testosterone levels are also observed after paradoxical sleep deprivation (PSD) which of course is a type of stress condition. This is in contrast to a marked presence of genital reflexes such as in penile erection and ejaculation in adult and old rats (Andersen et al., 2002; Andersen et al., 2004).

1.5.6 Oestradiol

Oestradiol or ooestradiol (E2 or 17 β-oestradiol) is a sex hormone. It is the predominant sex hormone present in females. It is also present in males, being produced as an active metabolic product of testosterone. It represents the major estrogen in humans. The ovary is the principal site of estrogen/ oestradiol production in females. Smaller amount is produced by the placenta, adrenal cortex and testes of men. Surprisingly, the immediate precursor of the female sex hormone is the male sex hormone, testosterone. During the reproductive years, most oestradiol in women is produced by the granulosa cells of the ovaries. Oestradiol, like other steroids, is derived from cholesterol, the parent of steroid hormones. After side chain cleavage and utilization of delta-5 pathway or delta 4-pathway, androstenedione is the key intermediate. A fraction of the androstenedione (Produced in the theca folliculi cells) is converted to testosterone which in turn undergoes conversion to oestradiol by an enzyme called aromatase. In an alternative pathway, androstenedione is aromatized to estrone, which is subsequently converted to oestradiol by 17 β-hydroxysteroid.

Oestradiol enters the cells freely and interacts with a cytoplasmic target cell receptor. After the estrogen receptor has bound its ligand, oestradiol can enter the nucleus of the target cell, and regulate gene transcription, which leads to formation of messenger RNA. The mRNA interacts with ribosomes to produce specific proteins that express the effect of oestradiol upon the target cell. Oestradiol binds well to estrogen receptors, ERα, and ERβ, in contrast to certain other estrogen, notably medications that preferentially act on one of these receptors. These medications are called selective estrogen receptor modulators or SERMs. Oestradiol is the most potent naturally occurring estrogen. In plasma, oestradiol is largely bound to sex hormones binding globulin and to albumin. Only a fraction of 2.21% is free and biologically active. The percentage remains constant throughout the menstrual cycle (Wu et al., 1976). Deactivation includes conversion to less active estrogens such as estrone and estriol. Estriol is the major metabolite. Oestradiol is conjugated in the liver by sulfate and glucouronide formation and such excreted via the kidneys. Some of the water soluble conjugates are excreted via the bile duct and partly reabsorbed after hydrolysis from the intestinal tract. This enterihepatic circulation contributes to the maintenance of oestradiol levels.

Oestradiol has a wide range of effects in both male and female reproductive system, fertility and body development. In the female, oestradiol acts as growth hormone for tissue of reproductive organs, supporting the lining of the vagina, the cervical glands, the endometrium, and the lining of the fallopian tubes. It enhances the growth of myometrium. Oestradiol appears necessary to maintain oocytes in the ovary. During the menstrual cycle, oestradiol that is produced by the growing follicle triggers, via a positive feed back system, the hypothalamic-pituitary events that lead to the luteinizing hormone surge, inducing ovulation. In the luteal phase, oestradiol in conjunction with progesterone, prepares the endometrium for implantation. During pregnancy, oestradiol increases due to placental production. In baboons, blocking of estrogen production leads to pregnancy loss, suggesting that oestradiol has a role in the maintenance of pregnancy. The role of estrogen in the process of initiation of labor is still being investigated. The development of secondary sex characteristic in women is driven by estrogens, to be specific, oestradiol. These changes are initiated at the time of puberty; most enhanced during the reproductive years, and become less pronounced with declining oestradiol support after menopause. Thus oestradiol enhances breast development, and is responsible for changes in the body shape, affecting bones, joints and fat deposition. Fat

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structure and skin composition are modified by oestradiol. The effect of oestradiol upon male reproductive system is also indispensable. Oestradiol is produced in the sertoli cells of the testes. There is evidence that oestradiol is designed to prevent apoptosis of male sperm cells (Pentikainen et al., 2006). Several studies have noted that sperm counts have been declining in many parts of the world and it has been postulated that this may be related to estrogen exposure in the environment (Sharp and Shakkebaek, 1993), therefore suppression of oestradiol production in a subpopulation of sub fertile men may improve semen analysis. It was equally noted that males with sex chromosome genetic conditions such as klinefelters syndrome will have a higher level of oestradiol. Oestrogen receptor (ERα) is essential for male fertility. Its activity is responsible for maintenance of epithelial cytoarchitecture in efferent ductules and the reabsorption of fluid for concentrating sperm in the head of the epididymis. This is achieved by its regulation of Na+/H+ exchanger-3 (NHE3) found in the caput and corpus epididymis, which is responsible for 90% luminal fluid reabsorption that thereby increases the concentration of sperm before entering the epididymis (Zhou et al., 2001).

Apart from its effects on the male and female reproductive system, oestradiol, influences other tissues or organs of the body. There is evidence that oestradiol has profound effect on bone. Individuals without oestradiol (or other estrogens) will become tall and eunuchoid as epiphysieal closure is delayed or may not take place. Bone structure is affected resulting in early osteopenia and osteoporosis ((Carani et al., 1997). Also post menopausal women, experience an accelerated loss of bone mass due to a relative estrogen deficiency. Oestradiol has also complex effects on the liver. It can lead to cholestasis. It affects the production of multiple proteins including lipoproteins, binding proteins, and proteins responsible for blood clotting. Estrogen can be produced in the brain from steroid precursors. As antioxidants, they have been found to have neuroprotective function (Behl et al., 1995). The positive and negative feed back loop of menstrual cycle involves oestradiol as a link to the hypothalamic-pituitary system to regulate gonadotropins. Estrogen is considered to play a significant role in women’s mental health, with links suggested between the hormone, mood and well being. Sudden drops or fluctuations in, or long periods of sustained low level of estrogen may be correlated with significant mood lowering. Clinical recovery from depression during postpartum, perimenopause and postmenopause was shown to be effective after levels of estrogen was stabilized and restored (Lasiuk and Hegadoren, 2007). Estrogen affects

48

certain blood vessels. They improve arterial blood flow and this has been demonstrated in colonary arteries (Collins, 1995). Estrogen is suspected to activate certain oncogenes, as it supports certain cancers, notably breast cancer and cancer of the uterine lining (Russo and Russo, 2006). In addition there are several benign gynecological conditions that are dependent on oestrogen such as endometriosis, leiomyomata uteri and uterine bleeding. The effect of oestradiol, together with estrone and estriol in pregnancy is less clear. They may promote uterine blood flow, myometrial growth, stimulate breast growth and at term, promote cervical softening and expression of myometrial oxytocin receptors. Oestradiol measurement could be done for medical uses to assess some of the effects for possible diagnosis and treatment. Serum oestradiol measurement in women reflects primarily the activity of the ovaries. As such, they are useful in the detection of baseline estrogen in women with amenorrhea or menstrual dysfunction and to detect the state hypoestrogenicity and menopause. Further more, estrogen monitoring during fertility therapy assesses follicular growth and is useful in monitoring the treatment. Estrogen producing tumors will demonstrate persistent high levels of oestradiol and other estrogens. In precocious puberty, oestradiol levels are inappropriately increased. In the normal menstrual cycle, oestradiol levels measure typically less than 50pg/ml at menstruation, rises with folicular development up to a peak of 200pg/ml and drop briefly at ovulation. It rises again during the luteal phase for a second peak. At the end of the luteal phase, oestradiol levels drop to their menstrual levels unless there is a pregnancy. During pregnancy, oestradiol levels rise steadily towards term. The source of these estrogen or oestradiol is the placenta, which aromatizes prohormones produced in the fetal adrenal gland. If oestradiol rise is obtained in males, it gives a signal for male infertility.

Sleep loss, has been associated with an alteration in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis, with stress hormone release and with autonomic activation. Animals, regardless of methods used to achieve sleep deprivation, have been observed during research investigations to have stressful experiences that result to alterations in catecholamine and hormones levels in addition to other physiological changes (Faroogui et al, 1996). During one of such work, oestradiol and estrone levels were observed to be reduced during paradoxical sleep deprivation (PSD) (Andersen et al., 2002). Oestradiol, testosterone and prolactin were strongly reduced during a 5 days rangers training course involving heavy physical activities and almost without sleep (Opstad and Aakvaag, 1982).

1.6 Body Weight

This is total mass or weight of a person in kilograms or pounds. Strictly this is measured without any anything on but practically it is measured with cloth on but without other heavy materials and accessories such as shoes, mobile phones and wallets. While the terms mass and weight are often used interchangeably, in the context of body weight, they refer to separate but related concepts in physics. Mass is a measure of an objects inatia and is independent on the effect of gravity while weight is a measure of force due to gravity. Thus if a person were to travel from earth to the moon where there is less gravity, his/her mass would remain unchanged but his or her weight would decrease.

The most practicable method of estimating ideal body weight is using the body mass index (BMI). BMI is calculated from measuring the body weight in kg and dividing it with the value of height in meter squared = Weight in kg Length in m2

A healthy body weight range is defined as BMI range of between 18.5 and 25. A BMI, below 18.5 is under weight, a BMI; between 25 and 30 is over weight. A BMI of 30 and 35 is obese and a BMI of 40 and above is morbidly obese. There are a number of factors that contribute to weight, such as

• Hereditary factors

• Hormonal abnormalities

• Lack of exercise

• Diet

• Life style.

Sleep deprivation has negative effect on body weight. In rats, prolonged or complete sleep deprivation, increased both food in take and energy expenditure with net effect of weight loss and ultimately death (Everson et al., 1989).

In human, the reverse is the case, following the report of many research studies. The increasing prevalence of weight gain, obesity and diabetes are being traced to the role of sleep loss among other factors. Current data suggest that the relationship between sleep restriction, weight gain and diabetes may involve three pathways; alteration in glucose metabolism, up regulation of appetite and decrease energy expenditure (Knusten et al., 2007).

Up regulation of appetite is followed by an increase in BMI, while insufficient sleep and the resulting sleepiness and fatigue may also have a lower level of energy expenditure than well-rested adult particularly in an environment that promotes physical inactivity. In one of the work done, it was seen that a feeling of hunger as as well as plasma ghrelin levels were elevated after one night of sleep deprivation whereas leptin concentration remained unaffected, thus providing further evidence for disturbing influence of sleep loss on endocrine regulation of energy which in long run may result in weight gain and obesity (Schmid et al., 2008).

Epidemiological data confirms that obesity accounts for about 6% of primary infertility and it may be surprising that low body weight in women accounts for 6% primary infertility. This 12% of primary infertility result from deviation in body weight from established norms, and that this infertility can be corrected by restoring body weight to within normal established limits. More than 70% of women who are infertile as a result of body weight disorder will conceive spontaneously if their weight disorder is corrected through a weight gaining or weight education diet as appropriate; yet body weight is often considered lastly in an infertility evaluation. It is therefore pertinent that the body weight of both partners of the infertile couple should be considered first when there is an obvious slender or obese body habitus in either partner (Green et al., 1988; Ates and Wentworth, 1982).

The mechanism for alteration of reproduction by body weight is biologically non ambiguous. Sex steroid hormones-testosterone (the principal male hormone) and oestradiol (the principal female hormone) are lipid soluble and thus accumulate in body fat. Once body fat stores are saturated with sex steroid hormone, they reach equilibrium with blood. In addition to the stored source of steroid hormone in the body fat, the gonads secrete testosterone and oestradiol to maintain the level necessary to sustain reproductive function. Also in addition to the storage capacity of the body fat for sex steroid hormones, adipose cells, convert the weak male hormone, androstenediol, to the weak female hormone, estrone. Estrone, though not as potent as oestradiol, has metabolic effect on the hypothalamus-pituitary axis of the brain (the area of the brain that regulates testicular and ovarian function) to alter reproductive function. These complex interactions have net effect of impairing reproductive function, the excess of androstenedione and oestradiol of overweight people, suppress FSH and subsequently cause infertility (Green et al., 1988).

1.7 Analysis of Methodology for Sleep Deprivation, Prion Protein and Hormones Assay

In science, sleep deprivation is used in order to study the functions of sleep and the biological mechanization underlying the effect to sleep deprivation. There are a number of sleep deprivation techniques among which are: Gentle handling method, single platform multiple platform, modified multiple, and the automated sleep deprivation. There are also the pendulum techniques for rodents; torture and pharmacological approach to human sleep deprivation.

1.7.1 Gentle handling

This often requires polysomnography during this sleep deprivation period. The animal and its polygraph records are continuously observed when the animal displays sleep electrophysiological posture. It is given an object to play with and activated by a caustic and if necessary tactile stimuli (Franken et al., 1991). This technique is used for total sleep deprivation as well as REM OR NREM sleep deprivation.

1.7.2 Single platform

This is probably one of the first scientific methods. During the sleep deprivation period, the animal is placed on an inverted flowerpot whose bottom diameter is small relative to animal size (usually 7cm adult rats.); the animal is able to rest on the pot and is even able to get NREM sleep. But at the onset of REM sleep, with its ensuring muscular relaxation, it would either fall into the water and climb back to its pot or would get it nose wet enough to weaken it (Rechtschaffen et al., 1999). This is used for REM sleep deprivation.

1.7.3 Multiple platforms

In order to reduce the elevated stress response induced by single platform method, this method was developed. The animals are placed into a large tank containing multiple platforms, thus eliminating the movement restriction experienced in the single platform, this technique is used for REM sleep deprivation only (van Hulzen and Coenen, 1981).

1.7.4 Modified Multiple Platforms

This is a modification of the multiple platform method where several animals together get the sleep deprivation.

1.7.5 Pendulum

Here animals are prevented from entering into the paradoxical (Ps) by allowing them to sleep for only brief period of time. This is accomplished by an apparatus, which moves the animal, cages backwards and forwards like a pendulum. At the extremes of the motion postural imbalance is produced in the animals forcing them to walk downwards to the other of their cages (van Hulzen and Coenen, 1981).

1.7.6 Automated sleep deprivation

Although, total sleep deprivation is frequently used in sleep researcher some of the techniques used such as gentle handling are labour consuming, and not standardized and boring. In order to minimize these limitations, an automated set up which can be used for total sleep deprivation is introduced. A shortfall of individual adjustable threshold of electromyogram (EMG) signals from sleep-deprived animals were used online to switch running wheels, incorporated into the home cages randomized direction of rotations, adaptable rotational sleep and automatic deactivation of the running wheels during quiet walking of the animals provided robust and standardized sleep deprivations with out increased stress when compared with gentle handling. These were used on line switch running wheel, during which walking of the animal provided robust and standardized sleep deprivation with out increased stress when compared with gentle handling. The set up can easily be introduced to a variety of home cages and is individually adaptable to each animal to sleep deprive (Fenzl et al., 2007).

1.7.7 Prion Protein Detection Method

Pathological Prion Protein implicated in transmissible spongiform encephalopathies, is detected by antibody based tests or bio essay to confirm the diagnosis of prion disease. Presently the western blot or an ELISA is officially used to screen the brainstem in cattle for the presence of PrPsc. The immuno polymerase chain reaction (IPCR), a technique whereby the exponential amplification ability of polymerase chain reaction (PCP) is coupled to the detection of protein by antibodies in a ELISA format, was applied in a modified real-time IPCR method to detect ultra low levels of prion protein using IPCR. Recombinant hamster PrPc was consistently detected at µg/ml. Proteinase K ( PK) digested scrapie infected hamster brain homogenates diluted to 10-8 (approximately 10-100 infectious units) was also detected with semi quantitative dose response. This level of detection is I million fold more sensitive than the level detected by western blot or ELISA and poses IPCR as a method capable of detecting PrPsc in the pre clinical phase of infection. However, it should be noted that unless complete Pk digestion of PrPc in biological materials is verified, ultra sensitive assays such as IPCR may inaccurately classify a sample as positive pathological sample (Barletta et al., 2005).

1.7.7.1 Western blot

The western blot (alternatively, immunoblot) is an analytical technique used to detect specific protein in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of polypeptide (denaturing conditions) or by the 3-d structure of protein (native/non-denaturing condition). The protein is transferred to a membrane (typical nitrocellulose or polyvinylidene diflouride (PVDF), where they are probed (detected) using antibodies specific to the target proteins. The process in this technique include sample preparation by humanization using homogenizer such as blender (for large sample volumes), smaller homogenizers (for smaller volumes) or by sonicattion, assorted detergent (e.g triton x 100), salt and buffers may be employed toencourage lysis of cells and solubilization of protein protease and phosphatase inhibitors are often added to prevent the digested of sample by its own enzymes. Tissue preparation is often done at cold temperature to avoid protein denaturation. The proteins of the sample are separated using gel electrophaorsis (e.g. sodium dodecyl sulfate (SDS) polyacrylmide gel electrophoresis). The separation of protein here may be by isoelectric point (PI), molecular weight, electric charger, or a combination of these factors. In order to make the proteins accessible for antibody detection, they are transferred from within the gel into a nitrocellulose or polyvinylidene fluoride (PVDF) where it should be stained with ponceau S for a check of uniformity of separation.

Blocking of nonspecific antibodies is done since the membrane has been chosen for its ability to bind protein and antibodies. This is achieved by placing the membrane in a dilute solution of protein, typically bovine serum albumin (BSA) or non-fat dry milk with a minute percentage of detergent such as tween 20, finally, the detection and analysis of the separation is done by Colorimetric, Chemiluminescent radioactive or fluorescence techniques (Renart et al., 1979; Neal Burnette, 1981).

For any of the methods, technique for extraction of protein from formalin-fixed tissues fro purposes of detection of PrP in formation tissues has been presented. It requires only minimal adaptation to existing Western blot procedures and could be incorporated into Western blot and ELISA existing protocol. This helps to alleviate some of the sample handling tissue and may prove for collection of field cases of TSE suspected animals and research work. Collected sample can now be placed immediately into 10% buffered formalin for a minimum of 2 weeks and up to 6 years. The specimen when required for further processing is brought out of the 10% buffered formalin and placed into 95% ethanol for 48 hours to remove the formalin. The tissues are then placed in phosphate buffered saline pH 7.5 for another 45 hours to remove the ethanol. At this point tissues are frozen at 20oC for later use or extracted for immediate use. Homogenization and further processing may depend on the actual techniques in use.

1.7.7.2 Immunohistochemistry

Immunohistochemistry process is another technique that is in use in the detection of prion disease. It refers to the process of detecting antigens (e.g. proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues (Ramos-Vera, 2005). Immunohistological staining is widely used in the diagnosis of abnormal cells such as those found in tumors, abnormal prion protein and other proteins in different parts of a biological tissue.

In this process, an antibody is conjugated to enzymes such as peroxidase that can catalyze a colour producing reaction. Alternatively, the antibody can also be tagged a fluorophore such as fluorescein or rhodamine. Proper tissue collection, fixation and sectioning is required depending on the purpose and thickness of the experimental sample. Because of the method of fixation and tissue preservation, the sample may require additional steps to make the epitopes available for antibody binding including deparaffinization and antigen retrieval: these steps often makes the difference between staining and no staining. Endogenous biotin or enzymes may need to be blocked or quenched, respectively prior to antibody staining, Triton X-100 are generally used in Immunohistochemistry to reduce surface tension, following less reagents to used to achieve and more even coverage of the sample. To reduce background staining in IHC the samples are incubated with a buffer that blocks the reactive sets to which the primary or secondary antibodies may otherwise blind. Common blocking buffers include normal serum, non-fat dry milk and bovine serum albumin (BSA) (Ramos-Vara, 2005). Haematoxylin and eosin are used as standards stains for detection of antibody reaction of PR sc in Immunohistochemistry.

1.7.7.3 ELISA

This is an immunological reaction where labeled antigens are allowed to complete with unlabelled antigens in a reaction with a limited quantity of antibodies. This type of competitive assay could be referred to as protein binding assay. This method of reaction is used for hormone assay as well as detection of abnormal prion protein in transmissible spongiform encephalopathies. It is heterogeneous non-isotopic assays that usually have an antibody immobilized into a solid support and the ligand is labeled with an enzyme. The enzymes used for this purpose must satisfy the following criteria:

(1) High specific activity: the signal amplification seen with an enzyme is related to the amount of substrate converted during the time of incubation capable of given the greatest amplification. Assay using such enzymes have excellent sensitivity and are able to detect very low concentration of ligand.

(2) The labels are stable during assays and under refrigerated storage conditions.

(3) The enzymes must not be present in the biological fluid or tissue sample to be analyzed.

(4) Must retain most of their activity when attached to the ligand or antibody (Stephen, 1989).

ELISA techniques for prion protein detection involve:

(a) providing a first solid support, comprising with a suitable peptide reagent.

(b) Contacting the first solid support with a sample under condition that allow pathogens prion protein when present in the sample, to bind to the reagent to form the first complex.

(c) Removing unbound sample materials.

(d) Dissociating the pathogenic prion from the first complex and

(e) Detecting the dissociated pathogenic prion using prion binding reagent (Celine et al., 2009).

1.7.7.3 Staining of Brain Tissue/Amyloid Proteins

The traditional way of identifying amyloid in tissue sections has been staining with congo red and demonstration of green birefringence under crossed polarizers, though haematoxylin and eosin (H and E) can also be used.

In this work congo red staining technique was used to stain the brain tissue of rats. Congo- red staining technique described by bennhold in 1922, has undergone several modifications to improve its sensitivity, specificity and reliability. The most common is the alkaline congo red method described by pucher and coworkers in 1962. Specificity is improved by using freshly prepared stain and a solution fully saturated with sodium chloride (Elghetany and Salem, 1989).

The other methodology involved in this research work were the special processing of rat killing called euthanization, a mercy killing of rodents as recommended by the Canadian council on animal care (CCAC, 1993) and the techniques of blood collection in rats as presented by van Herck (1998).

1.8 Nature of Research Subjects

A total of 32 albino rats were kept under observation for 5 days before subjecting them to research procedures. They were all assumed to be healthy and suitable for the nature of this research work since there were no symptoms of illness on them both in agility and physique. As a result of the research objective on the fertility hormone profiles of subjects, the sex status of the albino rats became a research interest too. 15 out of the 24 albino rats subjected to sleep deprivation were females while 9 were males. Emphasis was not given on the sex status of the 11 Albino rats used as control (non-sleep deprived rats) because they only helped in the comparison of changes from normal to abnormal prion protein in the sleep deprived rats. Such changes have no sex status significance. All the rats used were adult rats having a mean weight of 239g.

1.9 Consent

The subjects used in this research were animals: as a result, what was done was to follow the guideline of the Canadian council for animal care (CCAC, 1993).

1.10 Aim and Objectives of the Study

1.10.1 Aim of the Study

This work aims at determining the presence of PrP in Wistar albino rats and the possible changes that sleep deprivation can cause on Prion Protein and fertility hormones.

1.10.2 Specific Objectives of the Study

The specific objectives of this study were to:

(1) To determine if there is a change in the body weight of the rats after sleep deprivation.

(2) To determine the presence of PrP in Wistar albino rats

(3) To determine the effect of sleep deprivation on prion protein.

(4) To investigate whether there are changes in the serum status of FSH, LH, prolactin, Oesradiol, testosterone and TSH after sleep deprivation.

(5) To determine if there are changes in the morphology of the brain tissue of Wister albino rats after sleep deprivation.

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HOW TO RECEIVE PROJECT MATERIAL(S)

After paying the appropriate amount (#5,000) into our bank Account below, send the following information to

08068231953 or 08168759420

(1)    Your project topics

(2)     Email Address

(3)     Payment Name

(4)    Teller Number

We will send your material(s) after we receive bank alert

BANK ACCOUNTS

Account Name: AMUTAH DANIEL CHUKWUDI

Account Number: 0046579864

Bank: GTBank.

OR

Account Name: AMUTAH DANIEL CHUKWUDI

Account Number: 3139283609

Bank: FIRST BANK

FOR MORE INFORMATION, CALL:

08068231953 or 08168759420

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