Thesis Abstract

<p> 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) 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. <br></p>

Thesis Overview

<p> </p><p><strong>INTRODUCTION</strong></p><p>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).</p><p>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).</p><p>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).</p><p>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).</p><p>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).</p><p>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.</p><p><strong>1.1 Sleep</strong></p><p>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).</p><p><strong>1.1.1 Biology of Sleep</strong></p><p>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).</p><p><strong>1.1.2 Regulation of Sleep</strong></p><p>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).</p><p>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.</p><p><strong>1.1.3 Functions of Sleep</strong></p><p>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).</p><p>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).</p><p><strong>1.2 Sleep Deprivation</strong></p><p>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).</p><p><strong>1.2.1 Sleep Disorders</strong></p><p>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.</p><p><strong>1.2.2 Sleep Deprivation and Associated Problems</strong></p><p>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).</p><p><strong>1.2.3 Sleep Deprivation and Protein Metabolism</strong></p><p>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).</p><p>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).</p><p><strong>1.2.4 Sleep Deprivation and Prion Protein</strong></p><p>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).</p><p><strong>1.3 Prion Protein (PrP)</strong></p><p>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).</p><p>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.</p><p>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,<br>1990) and most PrP appear to be degraded in non-acidic compartments bound by cholesterol-rich membrane.</p><p>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)</p><p>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).</p><p>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).</p><p><strong>1.3.1 Functions of Prion Protein</strong></p><p>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.</p><p><strong>1.3.2 Prion Protein and Cell Membrane Viability</strong></p><p>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).</p><p><strong>1.3.3 Prion Protein and Sleep</strong></p><p>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).</p><p>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).</p><p><strong>1.3.4 Anti-Apoptotic Function</strong></p><p>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.</p><p>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).</p><p>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.</p><p>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).</p><p><strong>1.3.5 Protein and Immune System</strong></p><p>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).</p><p><strong>1.3.6 Prion Protein and Muscular Tone</strong></p><p>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.</p><p><strong>1.3.7 Abnormal Prion Protein</strong></p><p>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.</p><p>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.</p><p>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.</p><p><strong>1.3.8 The Pathogenicity of Prion</strong></p><p>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).</p><p>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).</p><p><strong>1.3.9 The Diseases of Prion (PrPres)</strong></p><p>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).</p><p>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).</p><p>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).</p><p>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.</p><p>‘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.</p><p>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).</p><p><strong>1.4 Endocrine System</strong></p><p>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.</p><p>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).</p><p>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).</p><p>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).</p><p>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).</p><p><strong>1.5 Hormones</strong></p><p>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.</p><p>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.</p><p><strong>1.5.1 Follicle Stimulating Hormone</strong></p><p>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 testi