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Stimulation and production of 11-deoxycortisol in the stress response of lamprey Roberts, Brent Warren William 2012

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STIMULATION AND PRODUCTION OF 11-DEOXYCORTISOL IN THE STRESS RESPONSE OF LAMPREY by Brent Warren William Roberts B.Sc. The University of Western Ontario, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (ZOOLOGY)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June, 2012  © Brent Warren William Roberts, 2012  Abstract This study is the first to provide direct physiological evidence that lamprey have a hypothalamus-pituitary-interrenal (HPI) axis that functions in a manner similar to that of more derived vertebrates. In teleost fishes, the hypothalamus produces corticotropinreleasing hormone (CRH), which stimulates the anterior pituitary to produce adrenocorticotropic hormone (ACTH), which in turn stimulates production of cortisol (F) from the interrenal cells of the head kidney. Although it has recently been shown that 11deoxycortisol (S) is the functional corticosteroid in lamprey, no studies have yet determined its mechanism of regulation or site of production. This study demonstrated that 1) exposure to acute stress by air-exposure caused plasma S concentrations to increase, supporting its role as a stress hormone; 2) intraperitoneal injections of lampreyCRH (0.1, 50, and 100 µg/kg) stimulated an increase in plasma S concentrations that was sensitive to dose, 3) intraperitoneal injection of four isoforms of lamprey-ACTH (each at 1.0 and 10 µg/kg) increased plasma S concentrations, although rates of production were low and varied by isoform between male and female subjects. Confirmation of the identity of 11-deoxycortisol was obtained by demonstrating that plasma extracts fractionated by high performance liquid chromatography (HPLC) had peaks of immunoreactive S that co-eluted with standard S using RIA. Finally, this study demonstrated that 4) lamprey mesonephric kidneys produced S in vitro when incubated in the presence of tritiated 17αhydroxyprogesterone (17αP), as well as in the absence of any precursor hormones. The identity of the tritiated S produced in vitro was determined by co-elution with standard S on HPLC, and confirmed by co-migration with standard and acetylated S on thin-layer ii  chromatography (TLC) after purification by HPLC. Together, these results provide supporting evidence that the stress response of lamprey, one of the oldest extant vertebrates, may be controlled through an axis similar to the hypothalamus-pituitaryinterrenal (HPI) axis of teleost fishes, although additional studies in this area will be required for further confirmation.  iii  Table of Contents Abstract .................................................................................................................................................... ii Table of Contents.................................................................................................................................. iv List of Figures ....................................................................................................................................... vii List of Abbreviations ........................................................................................................................... ix Acknowledgments................................................................................................................................ xi Chapter 1: General Introduction ..................................................................................................... 1 Thesis Introduction ................................................................................................................................... 1 Concept of Stress ........................................................................................................................................ 1 Three Stages of the Stress Response .................................................................................................. 4 The Hypothalamus-Sympathetic-Chromaffin Response ............................................................ 5 The Hypothalamus-Pituitary-Adrenal Response .......................................................................... 6 Evolution of the Stress Axis and its Molecules ............................................................................... 8 Hypothalamus-Pituitary-Interrenal Tissues ................................................................................... 8 Corticotropin-Releasing Hormone ...................................................................................................... 9 Adrenocorticotropic Hormone .......................................................................................................... 10 Corticosteroid Hormones .................................................................................................................... 11 Lamprey as a Model Species ............................................................................................................... 14 Thesis Objectives and Hypotheses ................................................................................................... 16 Chapter 2: Production and Regulation of 11-Deoxycortisol in the Stress Response of Lamprey.................................................................................................................................................. 18 Synopsis ...................................................................................................................................................... 18 Introduction .............................................................................................................................................. 19 Materials and Methods ......................................................................................................................... 21 Materials ......................................................................................................................................... 21 Collection and Maintenance of Animals.............................................................................. 22 Experimental Subjects ............................................................................................................... 22 Sampling Methods ....................................................................................................................... 22 Blood Collection ........................................................................................................................... 22 Tissue Sampling ........................................................................................................................... 23 Analytical Techniques ................................................................................................................ 23 iv  Radioimmunoassay .................................................................................................................... 23 Steroid Extraction........................................................................................................................ 24 Experimental Series 1: Acute Stress Treatment .............................................................. 25 Experimental Series 2: Corticotropin-Releasing Hormone Injections.................... 25 Confirmation of 11-Deoxycortisol ........................................................................................ 26 Experimental Series 3: Adrenocorticotropic Hormone Injections ........................... 26 Experimental Series 4: In Vitro Corticosteroidogenesis............................................... 26 Thin Layer Chromatography ................................................................................................... 27 High Performance Liquid Chromatography ...................................................................... 28 Steroid Acetylation and Analysis by TLC............................................................................ 29 Statistical Analysis ...................................................................................................................... 29 Results ......................................................................................................................................................... 30 Experimental Series 1: Acute Stress Treatment .............................................................. 30 Experimental Series 2: Corticotropin-Releasing Hormone Injections.................... 30 Confirmation of 11-Deoxycortisol ........................................................................................ 31 Experimental Series 3: Adrenocorticotropic Hormone Injections ........................... 32 Experimental Series 4: In Vitro Corticosteroidogenesis............................................... 32 Discussion .................................................................................................................................................. 33 Basal Stress Levels ...................................................................................................................... 34 Experimental Series 1: Acute Stress Treatment .............................................................. 35 Experimental Series 2: Corticotropin-Releasing Hormone Injections.................... 37 Confirmation of 11-Deoxycortisol ........................................................................................ 38 Experimental Series 3: Adrenocorticotropic Hormone Injections ........................... 39 Experimental Series 4: In Vitro Corticosteroidogenesis............................................... 41 Chapter 3: Conclusion ........................................................................................................................ 56 Summary .................................................................................................................................................... 56 Thesis Objectives and Hypotheses ................................................................................................... 57 Implications .............................................................................................................................................. 58 Limitations ................................................................................................................................................ 59 Future Directions .................................................................................................................................... 60 v  References ............................................................................................................................................. 63  vi  List of Figures Figure 2.1. Plasma 11-deoxycortisol concentration of adult male (n =3-5) and female (n = 4-7) Pacific lamprey after acute stress by air-exposure. Data are mean ± SE. Asterisks indicate a significant (* P < 0.05; ** P < 0.01; *** P < 0.001) difference relative to controls .................................................................................................................................................................. 44  Figure 2.2. Plasma 11-deoxycortisol concentration of adult male (n =6) and female (n =6) sea lamprey after intraperitoneal injection of lamprey corticotropin-releasing hormone (100 μg/kg body weight) or saline solution (0.90% NaCl). Data are mean ± SE. Asterisks indicate a significant (*P < 0.05; **P < 0.01; ***P < 0.001) difference relative to controls. ................................................................................................................................................................. 45  Figure 2.3. Plasma 11-deoxycortisol concentration of adult male (n =5) and female (n = 5) Pacific lamprey after intraperitoneal injection of lamprey CRH or saline solution (0.90% NaCl). Data are mean ± SE. Asterisks indicate a significant (*P < 0.05; **P < 0.01; ***P < 0.001) difference relative to controls. ....................................................................................................... 46  Figure 2.4. Plasma 11-deoxycortisol concentration from adult (A) male sea lamprey, (B) female sea lamprey, (C) male Pacific lamprey, and (D) female Pacific lamprey following fractionation by HPLC. Arrow shows the elution point of standard S........................................... 47  Figure 2.5. Plasma 11-deoxycortisol concentration of (A) adult male (n =5), and (B) female sea lamprey after injection with four isoforms of lamprey adrenocorticotropic-hormone or saline solution (0.90% NaCl) (59, 59P, 60, and 60P refer to the peptide length and phosphorylation state; C denotes saline-injected animals). Data are mean ± SE. Asterisks indicate a significant (*P < 0.05; **P < 0.01; ***P < 0.001) difference relative to controls. ................................................................................................................................................................. 48  Figure 2.6. Concentration of 11-deoxycortisol in tissues of (A) adult male (n=3) and (B) female sea lamprey after incubation in the presence or absence of four isoforms of lamprey ACTH. Data are mean ± SE. Asterisks indicate a significant (*P < 0.05; **P < 0.01; ***P < 0.001) difference relative to controls. ....................................................................................................... 49  vii  Figure 2.7. 3H counts (DPM) from TLC fractionation of incubation media from (A) male and (B) female sea lamprey mesonephric kidneys incubated with tritiated pregnenolone in the presence or absence of ACTH. Arrows show the migration points of standard 11deoxycortisol and pregnenolone. ................................................................................................................ 50  Figure 2.8. 3H counts (DPM) from TLC fractionation of incubation media (A) male and (B) female sea lamprey mesonephric kidneys incubated with tritiated progesterone in the presence or absence of ACTH. Arrows show the migration points of standard 11deoxycortisol and progesterone. ................................................................................................................. 51  Figure 2.9. 3H counts (DPM) from TLC fractionation of incubation media (A) male and (B) female sea lamprey mesonephric kidneys incubated with tritiated 17αOH-progesterone in the presence or absence of ACTH. Arrows show the migration points of standard 11deoxycortisol and 17αOH-progesterone. ................................................................................................. 52  Figure 2.10. 3H counts (DPM) of 17αP incubation media from (A) male and (B) female sea lamprey fractionated by 90 min HPLC runs. Arrows show the elution points of standard S and 17αP................................................................................................................................................................ 53  Figure 2.11. 3H counts (DPM) from TLC fractionation of male sea lamprey (A) control and (B) ACTH incubation media after purification with HPLC. Black bars represent the purified product of incubation, and grey bars represent the acetylated product. Arrows show the migration points of standard S and acetylated S. .................................................................................. 54  Figure 2.12. 3H counts (DPM) from TLC fractionation of female sea lamprey (A) control and (B) ACTH incubation media after purification with HPLC. Black bars represent the purified product of incubation, and grey bars represent the acetylated product. Arrows show the migration points of standard S and acetylated S. ............................................................... 55  viii  List of Abbreviations 17αP  17α-hydroxyprogesterone  1α-OH-B  1α-hydroxycorticosterone  ACTH  adrenocorticotropic hormone  ANOVA  analysis of variance  cAMP  cyclic adenosine monophosphate  CNS  central nervous system  CRH  corticotropin-releasing hormone  DOC  11-deoxycorticosterone  DPM  disintegrations per minute  ETOH  ethanol  F  cortisol  g  gram  GC  glucocorticoid  GnRH  gonadotropin-releasing hormone  HPLC  high performance liquid chromatography  MC  mineralocorticoid  MCR  melanocortin receptor  MEOH  methanol  MYA  million years ago  P  progesterone  %  percent  ix  Preg  pregnenolone  RIA  radioimmunoassay  S  11-deoxycortisol  SE  standard error of the mean  SNS  sympathetic nervous system  TLC  thin layer chromatography  x  Acknowledgments I would like to take this opportunity to thank everyone who helped me during my time spent in this program. Dr. David Close, for your continuing advice in the face of the somewhat risky line of research selected for this project. Especially for your help with the interpretation of the seemingly puzzling preliminary data that eventually led to the final set of experiments whose results are presented in this thesis. Dr. Colin Brauner, for your guidance and help maintaining focus on the important core aspects of my research. Your ability to almost immediately see the exact areas I struggled with, and then offer excellent advice was very much appreciated. Wes Didier, for your willingness to provide hands-on help for nearly every experiment I performed, especially the ones that extended to 14 hours or overnight. My family, especially my wife, who was subjected (most often involuntarily) to hours of discussion about comparative fish physiology and evolutionary endocrinology; you helped me reach conclusions about my results in increasingly shorter times. Campbell River Indian Band, for the continued sponsorship that allowed me to complete this program. I hope my work will provide encouragement to other band members considering graduate studies.  xi  Chapter 1: General Introduction Thesis Introduction The stress response in vertebrates involves physiological processes to restore homeostasis after contact with stressors. This response and the tissues, molecules, and mechanisms involved are highly conserved across taxa. Lampreys are the oldest extant vertebrates, and their physiology may be representative of the ancestral state of all vertebrates. However, whether lamprey have a hypothalamus-pituitary-interrenal (HPI) axis that functions in a manner similar to that of more derived vertebrates such as salmon is currently unknown. Determining whether this is the case has implications for our understanding of evolutionary physiology as well as for practical issues such as lamprey population control and environmental management practices. This thesis describes the first set of experiments that have been performed to determine whether lampreys respond to stress in the same way as more derived species. The Introduction that follows will review the field of stress from a historical context up to the current understanding and conclude with the specific objectives of this thesis.  Concept of Stress The concept of stress has been studied in a physiological context since the 19th century, when changes in the microscopic structure and chemical composition of organisms could first be accurately measured (Ottaviani and Franceschi, 1996). In 1878, Claude Bernard was the first to establish the concept of an internally-regulated environment in the body, which he called the “milieu interieur” (Cannon, 1929; Gross, 1  1998). While earlier scientists had recognized that organisms naturally made adjustments to maintain a vaguely defined “steady state”, Bernard’s idea was significant in that it specified that the living parts of a body existed in and depended upon the fluids that bathed them (Cannon, 1929). This fluid matrix had to be kept in balance when faced with external changes or challenges (Cannon, 1929; Galhardo and Oliveira, 2009). The full importance of Bernard’s work, however, was not appreciated until more than 50 years later, when Cannon (1924) demonstrated that secretions from the adrenal gland were crucial for maintaining the milieu interieur, an action he coined “homeostasis” as an expansion of Bernard’s original term (Cannon, 1929; Gross, 1998). The next major contributor to the field of internal regulation was Hans Selye, who is widely credited for integrating several seemingly unrelated studies and observations into the single unified concept of physiological stress (Selye, 1950; Esch et al., 1975; Lazarus et al., 1993; Ottaviani and Franceschi, 1996; Close, 2007). Until Selye’s discoveries in the second half of the 20th century, the term “stress” had not yet been used in biological contexts except where physical load or weight was being described (Selye 1956; Lazarus 1993). The central tenet of this new unified concept was that all living organisms are able to respond to stress, and that the basic pattern of reaction is always the same, regardless of the source of the stress. Selye’s first studies put forward the concept of a “general adaptation syndrome”, which described the morphological changes that occurred in animals during stages of alarm, resistance, and exhaustion (Selye, 1936; Selye, 1946). Animals continuously exposed to physical or chemical disturbances would initially develop resistance to them, but would eventually reach exhaustion and die (Selye, 1946). Selye would later label the disturbances as “stressors”, and the reaction of the organism “stress” 2  (Selye, 1950; Selye, 1956). Adding to Canon’s earlier work on adrenal secretions, Selye later provided important evidence that stressors activate the pituitary-adrenal axis to release corticosteroid hormones that bring about the stress response (Selye, 1950, 1956, 1973; Galhardo and Oliveira, 2009). Although a number of ideas put forward by Selye were either refuted or proven incorrect (Sayers, 1950; Munck et al., 1984; Sapolsky et al., 2000) the concept of corticosteroids being regulated through a stress axis had profound implications on the directions of future stress research. The present-day concept of stress emerged from the widespread adoption of specific terminology and a number of experiments in the 1980s that studied organisms’ abilities to acclimate to challenges (Mason, 1975; Selye, 1975; Mommsen et al, 1999). Alternatively described as “performance capacity” (Schreck, 1981), “allostatis” (Sterling and Eyer, 1988), or “energetic load” (Schreck and Li, 1991), the concept was expanded over the next decades to elucidate the link between the symptoms of stress and their physiological mechanisms and causes (Close, 2007). During this time, scientists were discovering that the effects of stress involved an increasing number of morphological and physiological changes that included weight loss, variations in body temperature, loss of eosinophil and white blood cells, alterations in the chemistry of body fluids, inflammation, and changes in the form and function of connective tissue, liver, kidney, as well as other tissues and organs (Ottaviani and Franceschi, 1996; Galhardo and Oliveira, 2009). Initially, it was generally assumed that powerful hormones such as glucocorticoids or biogenic amines were responsible for this wide variety of complex reactions and health complications (Ottaviani and Franceschi, 1996). However, the discoveries that the peptides and neurotransmitters involved in these systems had dual, triple, or even multiple roles 3  helped explain how infection and stress interacted with neuroendocrine responses to maintain homeostasis (Grossman and Roselle, 1983; Reichlin, 1993; Beishuizen, 2003). Eventually a consensus began to emerge that a number of biochemical cascades involving multiple sites of interaction among its systems were responsible for what is now understood to be the stress response (Shaw and Bush, 1966; Ottaviani and Franceschi, 1996; Herman et al., 2003; Galhardo and Oliveira, 2009 Goshen and Yirmiya, 2009; GadekMichalska and Bugajski, 2010). The key concept of the stress response is the multidirectional immuno-neuroendocrine interactions that maintain homeostasis under a variety of stress conditions (Reichlin, 1993; Besedovsky and del Ray, 1996; Bertok, 1998; Galhardo and Oliveira, 2009).  Three Stages of the Stress Response The stress response in fish is similar to that of other vertebrates, and the most recent framework used to describe this response considers three stages: primary, secondary, and tertiary (Barton, 2002; Iwama, 2007; Galhardo and Oliveira, 2009). The primary response engages two neuroendocrine axes: the hypothalamus-sympatheticchromaffin cell (HSC) axis, which stimulates production and release of catecholamines such as adrenaline and noradrenaline, and the hypothalamus-pituitary-interrenal axis (HPI), which stimulates production and release of corticosteroids such as cortisol (F) and corticosterone (B) (Galhardo and Oliveira, 2009). The secondary response involves physiological and behavioural adjustments such as changes in metabolism, blood and tissue chemistry, respiration, and cardiovascular and immune functions (Barton, 2002; Iwama, 2007; Galhardo and Oliveira, 2009). The tertiary response involves whole-animal 4  changes in growth and behaviour, with an eventual impact on the whole population (Barton, 2002; Iwama, 2007). It is also important to note that there are complex interactions between the stages, and that the primary and secondary responses may themselves directly affect secondary and tertiary responses without further input from external stressors (Barton, 2002).  The Hypothalamus-Sympathetic-Chromaffin Response The stress response begins when the sympathetic nervous system (SNS), activated through autonomic neurons running from the paraventricular nucleus of the hypothalamus to the locus coeruleus in the brainstem and spinal cord (Peter, 1990), detects a physical, chemical, or perceived stressor (Mazeaud et al., 1977; Reid et al., 1998). Physical stressors may include handling, capture, or confinement; chemical stressors may include low oxygen, acidification, or contaminant exposure; and perceived stressors include stimuli evoking the startle response or the presence of a predator (Barton, 2002). Through sympathetic nerve fibers and cholinergic receptors, the central nervous system (CNS) then innervates chromaffin cells (Sumpter, 1997), homologous to the adrenal medulla in mammals and located in various organs in different species, to stimulate production and release of catecholamines such as adrenaline and noradrenaline (Mazeaud et al., 1977; Mazeaud and Mazeaud, 1981; Reid et al., 1998). This reaction occurs in seconds, with secondary messenger cascades bringing about the “fight or flight” response: increased heart and respiration rate, increased blood pressure, and stimulation of glucose release, as well as depression of energy storage, feeding and digestion, and the inhibition of reproductive physiology (Mazeaud, et al., 1977; Peter, 1990; Reid et al., 1998; Sapolsky et al., 2000). 5  After the rapid activation of the hypothalamus-sympathetic-chromaffin (HSC) axis, blood concentrations of catecholamines decrease within minutes following contact with the initial stressor, although they can remain high and cause desensitization or impairment of the system if the stress becomes chronic (Schreck, 2000; Galhardo and Oliveira, 2009).  The Hypothalamus-Pituitary-Adrenal Response In response to the same stimulus, but delayed relative to the release of catecholamines, cytokines are produced by a variety of tissues. These cell-signalling molecules, especially interleukin-6 (IL-6), bind receptors in areas of the hypothalamus where there is no blood-brain barrier, such as the median eminence, and induce CRH transcription in the paraventricular nucleus of mammals, and the anterior preoptic area in other vertebrates (Imura et al., 1991; Spinedi et al., 1992; Theodosis and MacVicar, 1996; Szelenyi, 2001; Yao et al., 2008; Denver, 2009). Through positive feedback, CRH then stimulates production and secretion of adrenocorticotropic hormone (ACTH) from the anterior pituitary either by direct innervation (as in teleosts) or by being released through portal vessels to bind CRH type-1 receptors (other vertebrates) (Vale et al., 1981; Gorbman, 1995; Slominski et al., 2005; Gadek-Michalska and Bugajski, 2010). In agnathans, however, the structure of these tissues differs by having few or no blood vessels or neurons between the two regions and it is thought that hypothalamic regulation of the pituitary occurs by diffusion of CRH molecules across the connective tissue barrier (Nozaki, 2008). Cytokines do not directly stimulate production of ACTH, although catecholamines have been shown to do so in higher vertebrates. (Axelrod and Reisine, 1984; Gadek-Michalska and Bugajski, 2010). After stimulation by CRH, ACTH is produced within minutes (Assenmacher et al., 6  2006) and released into the blood, where it circulates and binds receptors on the adrenal zona fasciculate in mammals (Chrousos et al., 1985), and the interrenal cells in fish (Jones, 1962; Barton, 2002). The interrenal cells are generally located in the anterior portion of the kidney, and show wide morphological variation between taxonomic groups, although their microscopic structure is highly homogenous (Jones, 1962; Barton, 2002). Once ACTH binds membrane receptors, secondary messengers such as cyclic adenosine-monophosphate (cAMP) are used to stimulate synthesis and release of corticosteroids (Schimmer, 1995; Wendelaar Bonga, 1997; Mommsen et al., 1999). The corticosteroids then circulate to target cells, and because they are derived from cholesterol and are lipophilic, diffuse through membranes. Within target cells, they bind steroid hormone receptors which elicit positive or negative effects on target gene expression (Beato and Klug, 2000). The physiological results of these genomic actions become evident roughly one hour after contact with the initial stressor, and diminish within hours to days. Corticosteroids are the final products of the HPI stress response, and are responsible for restoring homeostasis by suppressing functions of the reproductive, inflammatory, and immune systems, while stimulating continued elevation of blood glucose levels through gluconeogenesis and inhibiting glucose storage (Sapolsky et al., 2000). Finally, corticosteroids inhibit secretion of CRH and ACTH, an action that is critical for negative feedback control of the HPI axis and avoiding stress-related pathologies resulting from chronic over-activity (Sapolsky et al., 2000; Korte et al., 2005; McEwen, 2006; Yao et al., 2008; Barr et al., 2009).  7  Evolution of the Stress Axis and its Molecules The HPI axis in fish is similar to the HPA axis in mammals and involves the same major tissues: the hypothalamus, the pituitary, and interrenal cells of the head kidney (homologous to the mammalian adrenal cortex). The molecules involved (CRH, ACTH, and corticosteroids) are also highly conserved and have been found in an extensive number of vertebrate and invertebrate species (Ottaviani and Franceschi, 1996).  Hypothalamus-Pituitary-Interrenal Tissues The evolution of the hypothalamus far predates that of the pituitary and the adrenal cortex / interrenal cells, and has been traced back to its origins as part of a simple brain with neurosecretory cells that existed in a common ancestor that vertebrates share with insects (Tessmar-Raible et al., 2007). The pituitary gland is thought to have evolved sometime in early chordate history, as it is present and structurally similar in all vertebrates, including agnathans such as hagfish and lamprey (Dores et al., 1984; Nozaki, 2008). It is proposed that the hypothalamus and pituitary began functioning together as a neuroendocrine regulatory axis before or during the differentiation of ancestral agnathans, and this two-way axis later incorporated adrenal-cortical tissue (Sower et al., 2009). The evolution and anatomical location of the ancestral adrenal-cortical tissue, however, has been a matter of controversy for over half a century and still remains unclear (Jones, 1962; Sandor, 1979; Weisbart and Youson, 1975; Close, 2007). Despite this uncertainty, it is possible that the ancestral vertebrate had incorporated the adrenal-cortical tissue into the HP axis as it has recently been shown that lamprey have a functional corticosteroid hormone, 11-deoxycortisol, and that intraperitoneal injection of human-CRH and lamprey8  pituitary extracts increase its concentration in plasma (Close et al., 2010). Furthermore, lamprey have a hypothalamus-pituitary-gonad axis, although sex steroids may have different roles in agnathans than in other fish (Bryan, 2003; Katz et al., 1982; Sower, 1998; Bryan et al., 2008). It has not yet been determined whether the response to stress in lamprey operates through an axis similar to the HPI axis of more recently-derived species.  Corticotropin-Releasing Hormone Corticotropin-Releasing Hormone (CRH) was first isolated by Vale et al. in 1981, and belongs to a family of neuropeptide transcription factors that are found throughout chordate taxa. They are related to diuretic peptides in insects as well as CRH-like molecules in other invertebrates, and are thought to have evolved from an ancestral precursor in a Precambrian metazoan (Ottaviani and Francesschi, 1996; Lovejoy and Balment, 1999; Huising and Flik, 2005; Barr et al., 2009). Chordate CRH is encoded on a single gene and has been highly conserved; the peptides are identical in humans and rats (Johnson et al., 1992; King and Nicholson, 2007). Although the structure of CRH has been conserved, its function has broadened as genome duplications have occurred throughout vertebrate evolution (Lovejoy and Balment, 1999). The ancestral functions of CRH likely included ion and metabolic regulation in homeostasis, while its involvement with the stress response is thought to have evolved much later with the chordata and the beginning of the hypothalamus-pituitary axis (Lovejoy and Jahan, 2006; Sower et al., 2009). It has been demonstrated that injection of human-CRH stimulates an increase of plasma 11deoxycortisol in lamprey (Close et al., 2010), although no studies have yet determined whether lamprey-CRH elicits a similar response. 9  Adrenocorticotropic Hormone Adrenocorticotropic Hormone (ACTH) was first isolated in 1943 by two independent groups (Li et al., 1943; Sayers et al., 1943). In vertebrates, the pituitary gland produces proopiomelanocortin (POMC), the precursor protein for the melanocortins, a group of hormones including ACTH, melanocyte-stimulating-hormones (MSH), and endorphins that are associated with the stress response and environmental acclimation (Mains et al., 1977; Dores et al., 1984; Takahashi et al., 2005). In gnathostomes, ACTH and MSH are encoded by a single gene, proopiomelanocortin (POMC), whose protein is posttranslationally modified into tissue-specific final products in the pars distalis (PD) and pars intermedia (PI) of the pituitary (Smith and Funder, 1988; Coste et al., 2002; Kawauchi and Sower, 2006). In lampreys, however, the two peptides are encoded by two independent genes; ACTH is encoded by the gene proopiocortin (POC) and is processed in the PD, while MSH is encoded by proopiomelanotropin (POM) and is processed in the PI. (Takahashi et al., 1995a/b; Kawauchi and Sower, 2006; Haitina et al., 2007). The ancestral gene encoding ACTH has been suggested to be POMC, and is thought to have been inherited differently among early vertebrates (Kawauchi and Sower, 2006). The lineage leading to agnathans underwent either an internal gene duplication or full genome duplication, giving rise to the two differentiated genes observed, while the lineage leading to gnathostomes inherited the single ancestral gene (Kawauchi and Sower, 2006; Haitina et al., 2007). ACTH and MSH bind specific melanocortin receptors (MCR), which diverged more than 400 MYA, likely before the duplication of POMC (Haitina et al., 2007). The MCR in lamprey may be sensitive to ACTH, as injection with homogenized lamprey-pituitary 10  increases plasma 11-deoxycortisol concentrations dose dependently (Close et al., 2010). Lamprey-MCR is also sensitive to both lamprey-MSH and mammalian-MSH (Haitina et al., 2007). However, injection of mammalian-ACTH does not stimulate production of 11deoxycortisol (Close, 2007). This is likely due to structural variance: human-ACTH is composed of 39-40 amino acids, while lamprey-ACTH is composed of 59-60 amino acids which undergo posttranslational phosphorylation at position 35, resulting in four unique peptides (Takahashi et al., 2006). There is currently no evidence for ACTH production in lamprey after exposure to acute stress.  Corticosteroid Hormones Corticosteroids are a group of vertebrate steroids that are produced and secreted primarily by the adrenal cortex in mammals, the adrenal gland in birds and reptiles, and the interrenal cells in fish, although recent studies have demonstrated their production in additional tissues in some mammals and birds (Bern, 1967; Lofts and Bern, 1972; Medhi and Sandor, 1972; Barton and Iwama, 1991; Feist and Schreck, 2001; Hess, 2007). In mammals, the adrenal cortex produces three major types of steroids in three internal zones. The zona fasciculate produces glucocorticoids, the zona glomerulosa produces mineralocorticoids, and the zona reticularis produces androgens. Glucocorticoids (GC) and mineralocorticoids (MC) make up the corticosteroids, and have similar molecular structures, stemming from a common evolutionary origin (Baker, 2003). Mineralocorticoids are characterized by hydroxyl groups at positions C11 and C21, and an aldehyde group at C18. Glucocorticoids also have a hydroxyl at position 21, but are further  11  characterized by the presence or absence of hydroxyl groups at C11 and C17 (Bern, 1967; Idler and Truscott, 1972; Sandor, 1972; Jones et al., 1972). The primary mineralocorticoid in mammals is aldosterone, which regulates ion exchange and water reabsorption (Norman and Litwack, 1997). Although there is no evidence of aldosterone production in fish, which is likely due to its complex synthesis, it was suggested that 11-deoxycorticosterone (DOC) functioned as the MC (Sturm et al., 2005). However, this idea was later refuted, and it has been shown that cortisol functions both as a GC and MC in fish (McCormick et al., 2008). Glucocorticoids are the final products of the HPI axis which restore homeostasis by effecting a number of physiological changes (Charmandari et al., 2005). They function as transcription factors by binding receptors and being transported to the nucleus where they positively or negatively affect gene expression (Sapolsky et al., 2000; Habib et al., 2002). The primary glucocorticoids are cortisol and corticosterone in mammals (Bern, 1967; Idler and Truscott, 1972); corticosterone in amphibians, reptiles, and birds (Sandor 1971); and cortisol in more derived fish (Idler and Truscott, 1972). The only known exception is 1αhydroxycorticosterone (1α-OH-B), which is produced by elasmobranchs (Idler and Truscott, 1972). The actions of corticosteroids have been conserved throughout vertebrate evolution, but until recently, it was unclear what the ancestral corticosteroid was. While early literature reports the presence of cortisol and corticosterone in both hagfish and lamprey, the concentrations vary more than a thousand-fold between papers (Phillips et al., 1962; Buus and Larsen, 1975; Weisbart et al., 1980; Adams et al., 1987) and the findings have not 12  been substantiated or confirmed since. This variation may have been caused by three possible factors. First was the use of early assay techniques and technology; several studies were performed when the technology and techniques were in early stages (Zvi, 2007). Second, despite the high sensitivity of radioimmunoassay (RIA) techniques, the low concentrations of hormones found in these species compared to teleosts may have resulted in the misinterpretation of background levels for significant results. Third was potential biochemical cross-reactivity, as molecules that vary minimally in structure have been shown to bind the same antibodies and result in false positives (Yalow and Berson, 1960; Tate and Ward, 2004). Although Close et al., (2010) recently determined that 11-deoxycortisol is the primary corticosteroid in lamprey and may likely be the ancestral corticosteroid hormone for all vertebrates, no studies have yet determined its site or method of production. Production of corticosteroids in vertebrates is found in specialized adrenocortical tissues such as the adrenal cortex in mammals and the interrenal cells of the head kidney in fish (Barton and Iwama, 1991; Bern, 1967; Bush and Willoughby, 1957; Feist and Schreck, 2001; Schmidt and Soma, 2008; Schmidt et al., 2009). However, a number of studies have shown that different vertebrates produce corticosteroids in a wide variety of cells such as skin cells in mammals; immune tissue and brain in birds (Schmidt and Soma, 2008) and gonads in salmon, (Fitzpatrick et al., 1986; Ueda et al., 1983) rats, (Grgurevic et al., 2008), and lamprey (Weisbart and Youson, 1977; Weisbart et al., 1978; Kime and Callard, 1982; Sower 2009). Because of this variation, it is currently unknown where the ancestral vertebrate produced the corticosteroids central to the stress response, although it is known that the most basal extant vertebrates with completely differentiated tissues able to produce 13  corticosteroids are the elasmobranchs (Denver, 2009). Determining which tissue is responsible for production of the corticosteroids used in the stress response in lamprey will provide insight into the evolution of the stress response in vertebrates.  Lamprey as a Model Species Lampreys belong to the superclass Agnatha, which split from the rest of the vertebrates sometime between 564 million and 525 million years ago (MYA) (Forey and Janvier, 1993; Kumar and Hedges, 1998; Hedges and Kumar, 2003; Bryan, 2004). They are the earliest known vertebrates, and occupy a unique phylogenetic position that is widely considered to closely represent the ancestral state of all vertebrates (Thornton, 2001; Kuratani et al., 2002; Baker, 2004; Sower et al., 2009; Close et al., 2010; Smith et al., 2010). While the exact phylogenetic relationship between gnathostomes, extant agnathans, and the ancestral agnathan are a matter of some controversy, what is certain is that all extant vertebrates evolved from one ancestral agnathan (Mallatt and Sullivan, 1998; Kuratani et al., 2002). Developmental mechanisms that are present in both lamprey and gnathostomes can be considered to have existed in the ancestral agnathan, while developments present only in gnathostomes can be considered to have evolved after the lineages split (Kuratani, et al., 2002). Of interest to this study is that lampreys are the first to have evolved a stress hormone with biological activity similar to that of more derived vertebrates (Close et al., 2010). Accordingly, understanding the physiology and mechanisms of their stress response can have important implications for the way we understand the evolution of the stress response in all vertebrates (Bryan, 2004).  14  The phylogenetic relationships between individual lamprey species were initially determined by comparisons of their dentition, although a more recent study analyzing 32 morphological characteristics has revealed a new phylogenetic tree (Hubbs and Potter, 1971; Potter, 1980; Potter and Hillard, 1987; Gill et al., 2003). It appears that Ichthyomyzon is the ancestral genus of all lampreys in the northern hemisphere, and Ichtyomyzon unicuspis (silver lamprey) is agreed to be the extant ancestral species (Gill et al., 2003). The genus Petromyzon, in which P. marinus (sea lamprey) is the only species, is derived from Ichthyomyzon, and the two are considered to have monophyletic origins (Gill et al., 2003). The genus Lampetra, which includes L. tridentata (Pacific lamprey), is the most recently evolved genus. Most studies of lamprey in North America have focused on sea lamprey, which is likely a result of their overabundance in the Great Lakes and their impact as an invasive species since their introduction in or before the 1930s (Coble et al., 1990; Hansen, 1996; Bryan et al., 2006). However, other species are in decline worldwide, including those used as a traditional food source for Native Americans as well as in Europe (Renaud, 1997; Maitland, 1980; Almeida et al., 2000; Close et al., 2002). Further understanding of the biology of the lamprey stress response is therefore desirable because of its potential use as a clinical indicator for fisheries managers, both for population control in the Great Lakes, and for conservation practices on the West coast.  15  Thesis Objectives and Hypotheses The overall objective of this thesis was to determine whether lamprey have a functional hypothalamus-pituitary-interrenal (HPI) axis similar to that of more-derived teleost fishes. Specifically: Objective 1: To determine whether plasma 11-deoxycortisol concentrations will increase following acute stress by air-exposure in Pacific lamprey. Hypothesis 1: Plasma 11-deoxycortisol concentrations will increase 1 h following acute stress by air-exposure. Objective 2: To determine whether intraperitoneal injection of sea lamprey-CRH will stimulate an increase in plasma 11-deoxycortisol concentrations in sea lamprey and in Pacific lamprey. Hypothesis 2: Intraperitoneal injection of lamprey-CRH will increase plasma 11deoxycortisol concentrations 1 h following injection. Objective 3: To determine whether intraperitoneal injection of the four isoforms of sea lamprey-ACTH will stimulate an increase in plasma 11-deoxycortisol concentrations in sea lamprey. Hypothesis 3: Injection of all four isoforms of lamprey-ACTH will increase plasma 11deoxycortisol concentrations 1 h following injection.  16  Objective 4: To determine the site of production for 11-deoxycortisol in sea lamprey by in vivo incubations in the presence and absence of tritiated precursor hormones. Hypothesis 4: 11-deoxycortisol will be produced by the mesonephric kidneys.  17  Chapter 2: Production and Regulation of 11-Deoxycortisol in the Stress Response of Lamprey Synopsis The primary objective of this study was to characterize the stress response in lamprey by measuring increases in plasma 11-deoxycortisol (S) concentrations after exposure to acute stress by air-exposure, and in response to intraperitoneal injections of lamprey-CRH1-41, and one of four isoforms of lamprey-ACTH (ACTH1–59, ACTH1–59; 35P, ACTH1–60, and ACTH1–60; 35P). Exposure to acute stress caused plasma levels of S to rise by 1 h, supporting its role as a stress hormone in Pacific lamprey. Intraperitoneal injection of lamprey-CRH resulted in significant increases of S that were sensitive to dose, demonstrating for the first time its effectiveness in lamprey. Confirmation of the identity of S was obtained by demonstrating that plasma extracts from lamprey that were injected with CRH, fractionated by HPLC had peaks of immunoreactive S that co-eluted with standard S. Injection of each of four lamprey-ACTH isoforms resulted in significant levels of S, although rates of production were low and varied by isoform between male and female animals. This study also demonstrated that lamprey mesonephric kidneys were able to produce S in vitro when incubated in the presence of tritiated 17αP, as well as in the absence of any precursor hormones. Identification of the tritiated product was confirmed by co-elution with standard S on HPLC, and by co-migration with HPLC-purified standard and acetylated S on TLC.  18  The combined results provide supporting evidence that the stress response of lamprey is controlled through an axis similar to the hypothalamus-pituitary-interrenal (HPI) axis of teleost fishes.  Introduction Lampreys have been the focus of study by wildlife managers and evolutionary and comparative biologists since the early- to mid-twentieth century. After their introduction as an invasive species to the Great Lakes in the 1930s, sea lampreys have been the subject of study by wildlife management programs seeking to mitigate their impact on native species (Coble et al., 1990; Hansen, 1996). More recently, evolutionary and comparative biologists have recognized lamprey as valuable due to their unique phylogenetic position that may provide insight into the biology of the ancestral vertebrate (Hedges and Kumar, 2003; Kuratani et al., 2002). Recent progress has been made into a number of areas of lamprey biology including their lifecycle, physiology, and biochemistry. A recent study into the stress response in lamprey by Close et al. (2010) identified 11-deoxycortisol as a functional corticosteroid that may be representative of the ancestral stress hormone. Following the characterisation of this hormone, it is possible to test the hypothesis that the HPI axis in lamprey is functional or similar to that of more derived vertebrates such as teleosts. In teleost fishes, the HPI axis is stimulated after exposure to a physical, chemical, or perceived stressor, which causes CRH to be produced by the hypothalamus (Barton, 2002). CRH then stimulates the anterior pituitary to produce ACTH. This occurs by direct innervation in teleosts, and by diffusion of CRH molecules across membranes in agnathans 19  (Nozaki et al., 1994; Nozaki et al. 2008). ACTH is then released into circulation and stimulates the interrenal cells of the head kidney to produce cortisol. This system of response to stress is similar to the HPA system of mammals, and has been conserved in most vertebrates. CRH is a 41 amino acid peptide belonging to a family of neuropeptide transcription factors produced in the hypothalamus that have been highly conserved (Ottaviani and Francesschi, 1996; King and Nicholson, 2007). Although Close et al. (2010) demonstrated that injection of human-CRH stimulated an increase in plasma S concentrations, such an effect has not yet been shown with lamprey-CRH. Due to the high similarity, however, it is likely that such a response is possible. Once CRH is produced by the hypothalamus, it stimulates production of ACTH in the anterior pituitary. In teleost fishes stimulation of ACTH by CRH occurs by direct innervation, but in agnathans, the tissues are structurally different and it is thought that this occurs by diffusion across the connective tissue barrier (Nozaki et al., 1994; Nozaki, 2008). Furthermore, while gnathostome ACTH is composed of 39-40 amino acids, lamprey ACTH is composed of 59-60, with posttranslational phosphorylation occurring at position 35 in two of the four isoforms (Takahashi et al., 2006). This modification results in four unique isoforms that are each significantly different than the single peptide found in most vertebrates. Since the discovery of these four isoforms, no published data exist on their effect on lamprey physiology. Additionally, no form of ACTH has been shown to stimulate production of 11-deoxycortisol, nor is there currently any evidence for ACTH production in lamprey after exposure to acute stress. The study by Close et al. (2010), however, 20  demonstrated that intraperitoneal injection of lamprey pituitary extract stimulated dosedependent production of S, indicating that a product of this tissue may be involved in the stress response. After release from the pituitary, ACTH enters the circulation and stimulates the interrenal cells of the head kidney to produce and secrete the glucocorticoid cortisol, which is in turn released into the circulation to reach target tissues (Barton, 2002). The interrenal tissue is homologous to the adrenal cortex of mammals, and although its morphology varies widely across taxonomic groups, it is often generally found in the anterior kidney (Barton, 2002). As lampreys metamorphose, however, the anterior portion of their kidney degenerates, leaving only the mesonephric and posterior areas intact (Youson, 1970). It is not known whether any section of the lamprey kidney is able to produce glucocorticoids, nor is there evidence for their production in any other tissue either in vitro or in vivo. The objectives of this study were 1) to determine whether plasma S concentrations increase in response to acute stress by air-exposure in adult lamprey, 2) to determine whether lamprey-CRH would stimulate an increase in plasma concentrations of S, 3) to determine whether lamprey-ACTH would increase plasma S concentrations, and 4) to determine which tissue(s) produce S.  Materials and Methods Materials Radiolabeled steroids were purchased from American Radiolabeled Chemicals (St. Louis, MO, USA). Synthetic steroids, the antibody to 11-deoxycortisol, and all other 21  chemicals and reagents were purchased from Sigma (Sigma Aldrich Chemical Co., St. Louis, MO, USA) unless otherwise noted. Collection and Maintenance of Animals Adult Pacific lampreys (Lampetra tridentata) were collected by net in the fish ladder at Stamp River Falls, British Columbia in June 2010. Adult sea lampreys (Petromyzon marinus) were obtained from the Sea Lamprey Control Program at the Department of Fisheries and Oceans in Sault Ste. Marie, Ontario in July 2010 and July 2011. Animals were transported to the University of British Columbia, Vancouver, BC, where they were held at 4-6°C in covered, insulated tanks filled with dechlorinated tap water from the City of Vancouver. Sea lampreys and Pacific lampreys were approved for use in these experiments, which were performed according to the University of British Columbia Animal Care protocol A11-0055. Experimental Subjects For all experiments, adult lampreys were acclimated in covered, insulated, flowthrough tanks (254 L) filled with dechlorinated tap water from the City of Vancouver at 1012°C for at least one week before experiments. Sampling Methods Blood Collection In Experimental Series 1, 2, and 3 described below, once fish were anesthetized, fish were placed upside down in a plastic trough and blood was collected by cardiac puncture 22  using Vacutainers coated with EDTA to prevent clotting (Becton Dickinson-Canada, Mississauga, ON, Canada). Samples were immediately placed on ice. Fish were placed in a freshwater recovery bucket and then returned to holding tanks for recovery. Total sampling time for each tank did not exceed three minutes. Blood samples were centrifuged for 12 min at 2500 RPM and 4°C (Beckman Coulter). Plasma was frozen at -80°C until RIA was conducted. Tissue Sampling Following acclimation, fish to be euthanized were netted out and immediately immersed in an overdose of anesthetic solution (0.2 g/L – 0.3 g/L of MS-222; Argent Chemical Laboratories, Inc.). At this dose, most movement stopped within one minute and death occurred within 2 - 3 minutes. Euthanized animals were placed on a surgery table, and then tissues including kidneys, gills, gonads, and livers were collected and immediately placed in L-15 incubation medium (Sigma-Aldrich) on ice. Animal remains were bagged, labeled, and disposed of according to UBC policy. Analytical Techniques Radioimmunoassay Radioimmunoassays (RIAs) were performed as in Scott et al., (1980). Briefly, RIAs were conducted in duplicate in 10 mm x 75 mm glass culture tubes (Fisher Scientific). Nine standards, also in duplicate, were made up over the range 500 – 1.95 pg/100 L tube. Unknown sample tubes contained a total volume of 100l, made up of 20 L plasma and 80 L assay buffer (50 mM sodium phosphate, pH 7.4, 0.2% BSA, 137mM NaCl, 0.40 mM EDTA, 23  and 0.77 mM sodium azide). Binding reagent was made by adding radiolabel and antibody such that when 100 L was dispensed to all tubes, each tube would contain 5000 disintegrations per minute (DPM), and in the absence of any standard steroid, 50% of the radiolabel would be bound to the antibody. Blank tubes with no antibody, and tubes necessary to determine the total and maximum DPM counts were also included. All tubes were incubated at 4°C overnight, separated with 500 mL of charcoal solution at 0°C (50 mM sodium phosphate, pH 7.4, 0.1% gelatin, 1.0% dextran-coated charcoal), centrifuged at 2500RPM, 4°C for 12 min, decanted into 7ml scintillation vials, and mixed with 5ml scintillation cocktail. DPM were counted with an LS-6500 (Beckman Coulter) scintillation counter. Steroid Extraction Steroid extraction was performed as in Newman et al. (2008). Briefly, Sep-paks were activated with 5 mL methanol (MEOH) and rinsed with distilled water (ddiH2O). Supernatant was pushed through 0.45 μm filters (Millipore, Billerica, MA, USA) and loaded onto Sep-Paks (Waters, Milford, MA, USA), which were rinsed with 5 mL ddiH2O and then eluted with 5 mL MEOH. Samples were then loaded onto a CentriVap Concentrator (Labconco, Kansas City, MO, USA) to evaporate MEOH elute overnight. To each dried tube, 1 mL ETOH was added, mixed, and pipetted into microfuge tubes, which were labeled and frozen at -80°C until needed for analysis.  24  Experimental Series 1: Acute Stress Treatment Adult Pacific lampreys were subjected to acute air exposure. Pacific lampreys were netted out of the control tanks, placed in a dry bucket for 1 min, and then returned to a recovery tank. Lampreys were then anesthetised with MS 222 (0.1 – 0.15 g/L) and sampled at 5 min, 10 min, 60 min, 8 h, and 24 h following air-exposure (n = 3 - 7). Control fish (n = 3 - 4) were left in the holding tanks undisturbed until they were anesthetised and sampled. Experimental Series 2: Corticotropin-Releasing Hormone Injections Adult sea lampreys were injected with either corticotropin-releasing hormone or saline solution (0.90% NaCl). Sea lampreys were injected intraperitoneally with corticotropin-releasing hormone (CRH1-41), based on the sequence identified from the sea lamprey genome database (with the help of Scot Libants at Michigan State University), and custom synthesized by New England Peptide (Gardner, MA, USA). Following acclimation, fish were netted out of tanks, immediately immersed in anesthetic solution, and once anesthetized, placed upside down in a plastic trough. CRH dissolved in saline solution (0.90% NaCl) was injected intraperitoneally at doses of 0.1 μg/kg, 50 μg/kg, or 100 μg/kg. Saline solution (0.90% NaCl) was used as a control. Once treated, fish were placed in a freshwater recovery bucket and then returned to holding tanks. Time spent for each set of injections did not exceed three minutes per tank.  25  Confirmation of 11-Deoxycortisol Steroids were extracted from the plasma of both sea lamprey and Pacific lamprey in the Series 2 Experiments and were fractionated by HPLC and re-assayed with S antibody as previously described by Close et al. (2010). Experimental Series 3: Adrenocorticotropic Hormone Injections Adult sea lampreys were injected with either adrenocorticotropic hormone, or saline solution (0.90% NaCl) solution. Sea lampreys were injected intraperitoneally with one of four isoforms of lamprey adrenocorticotropic hormone (ACTH1–59, ACTH1–59; 35P, ACTH1–60, and ACTH1–60; 35P), based on the sequences published by Takahashi et al. (2006), and custom synthesized by Bachem (Torrance, CA, USA). Following acclimation, fish were netted out of tanks, immediately immersed in anesthetic solution, and once anesthetized, placed upside down in a plastic trough. The four isoforms of ACTH were dissolved in saline solution (0.90% NaCl) and injected at doses of 1.0 μg/kg or 10 μg/kg. Saline solution (0.90% NaCl) was used as a control. Once treated, fish were placed in a freshwater recovery bucket and then returned to holding tanks. Time spent for each set of injections did not exceed three minutes per tank. Experimental Series 4: In Vitro Corticosteroidogenesis The objective of the first incubation experiment was to determine which, if any, of the kidney, gonads, gill, or liver were able to produce 11-deoxycortisol. Also tested in this experiment was whether the rate of production of 11-deoxycortisol would be affected by  26  the presence or absence of ACTH, as Experimental Series 3 determined that certain isoforms of ACTH significantly raised plasma levels of 11-deoxycortisol in vivo. Tissue samples were weighed, diced with a razor blade, and placed in 15mL conical tubes containing 5mL of L-15 incubation media (Sigma-Aldrich) on ice. Either all four isoforms of ACTH (ACTH1–59, ACTH1–59; 35P, ACTH1–60, and ACTH1–60; 35P; Bachem) at a dose of 100 ng/ml or an equivalent volume of saline solution (0.90% NaCl) were added to tubes. The conical tubes were sealed, placed horizontally, and incubated at 10°C for 4 h at a slow but constant shaker speed. After incubation, tubes were centrifuged at 2500RPM, 4°C for 12 min. After results from the first incubation experiment were obtained, the objective for the second incubation experiment became to determine which precursor steroid(s) the kidneys were able to convert to 11-deoxycortisol. The procedure was the same as above, but 1 Ci – 2 Ci of 3H-progesterone, 3H-pregnenolone, or 3H-17α-hydroxyprogesterone (American Radiolabeled Chemicals) were added to each incubation tube. Thin Layer Chromatography In order to determine whether any of the radiolabeled precursor steroids were converted to 11-deoxycortisol during the second, radioactive incubations, an initial analysis of the products was performed by thin layer chromatography (TLC). Volumes corresponding to 10,000 DPM – 40,000 DPM of extract were placed in 10 mm x 75 mm glass culture tubes (Fisher Scientific) containing 10 l of standard steroids. These were dried down under nitrogen at 40°C, resuspended in 100 l ethyl acetate, and loaded onto 27  separate lanes of pre-coated silica-gel TLC plates (Whatman Inc. Piscataway, NJ, USA). The plates were developed for 30 min with chloroform/ethanol/acetic acid (50/50/0.002 v/v/v) after equilibrating for 30 min. The positions of standard steroids were noted by placing the plates under a UV source. The lanes were divided into 4 mm sections, scraped off into scintillation vials, mixed with 5 mL SafetySolve scintillation cocktail (Research Products International Corp., Mount Prospect, IL, USA) and DPM were counted with an LS6500 (Beckman Coulter) scintillation counter. High Performance Liquid Chromatography Once the initial analysis indicated which precursors were converted to 11deoxycortisol, products of the appropriate incubation media were purified by HPLC. Volumes corresponding to 15,000 DPM of extract were mixed with 20 g of standard steroids, dried down under nitrogen at 40°C, resuspended in 1 mL acetonitrile/water/formic acid (30/70/0.01, v/v/v), centrifuged at 14,000 RPM for 10 min, and then loaded onto a C18 reverse-phase HPLC column (Alltima, 4.6 mm x 250 mm, Alltech, Dearfield, IL, USA) fitted with a guard module. The solvents used to create the column gradient were 0.01 % formic acid (solvent A) and 70 % acetonitrile (solvent B) and were developed as follows: 0-10 min: 28 % B; 10-60 min: 28-100 % B; 60-90min 100 % B. Total development time was 90 min. Fractions were collected every 1 min between 20 min and 75 min in 16 mm x 100 mm culture tubes (Fisher Scientific). UV absorptions of eluate were monitored and recorded with a photodiode array detector (Shimadzu) to determine positions of standard steroids. Once collected, samples from each fraction were mixed with  28  5 mL scintillation cocktail (RPI Corp.) and DPM were counted with an LS-6500 (Beckman Coulter) scintillation counter. Steroid Acetylation and Analysis by TLC Steroid acetylation and analysis by thin-layer chromatography was performed as in Bryan et al., (2004). Briefly, volumes containing 100,000 DPM of 3H-11-deoxycortisol were fractionated on HPLC as described above. Part of the fractions (250 L containing 50,000 DPM) corresponding to the elution position of S were placed in a 16 mm x 100 mm culture tube (Fisher Scientific) containing 10 g of standard S. The solvents were removed, replaced by 100 L pyridine and 100 L acetic anhydride, covered, and left overnight at room temperature. The remaining 250 L of the same fractions were mixed with 10 g of standard S in a separate glass tube. The solvents in both tubes were evaporated and replaced with 100 L of ethyl acetate. These were loaded on to separate lanes of a TLC plate, which was developed for 30 min with chloroform/ethanol/acetic acid (50/50/0.002 v/v/v) after equilibrating for 30 min. The positions of standard steroids were noted by placing the plates under a UV source. The lanes were divided into 4 mm sections, scraped off into scintillation vials, and mixed with 5 mL scintillation cocktail (RPI Corp.). DPM were counted as above. Statistical Analysis Data are expressed as mean ± SE, and statistical significance is assumed at P<0.05. Statistical analyses were performed using Prism 5.00 (GraphPad Software Inc, California, USA). For Experimental Series 1 and 4, one way ANOVA followed by Tukey’s comparison, as 29  in Grutter and Pankhurst (2000), were used to assess differences. For Experimental Series 2 sea lamprey CRH injections, a two-tailed Student T test was used to assess the differences as in Close et al. (2010). For Experimental Series 2 Pacific lamprey CRH injections and Experimental Series 3 sea lamprey ACTH injections, significant differences between means were evaluated using one way ANOVA followed by Dunnett’s comparisons as in Bryan et al. (2004). Males and females were analyzed separately in all cases.  Results Experimental Series 1: Acute Stress Treatment Exposure to acute stress by air-exposure was found to significantly (P<0.001) increase plasma concentrations of 11-deoxycortisol after one hour in both male (n = 3-5) and female (n = 4-7) Pacific lamprey relative to time 0 (Fig 2.1 A / B). Plasma sampled at 1 h after air-exposure contained 6.7 ± 1.2 ng/ml (mean ± SE) of S in males and 4.3 ± 1.0 ng/ml of S in females, compared to 1.6 ± 0.49 ng/ml and 1.01 ± 0.06 ng/ml in males and females sampled immediately after air-exposure, respectively. No significant differences were found in fish sampled at 5 min (0.97 ± 0.15 ng/ml males; 1.3 ± 0.06 ng/ml females) or at 10 min (0.83 ± 0.08 ng/ml males; 0.98 ± 0.11 ng/ml females). Levels of S had returned to basal levels by 8 h (1.2 ± 0.02 ng/ml males; 1.7 ± 0.23 ng/ml females) with no further observed changes at 24 h (1.3 ± 0.15 ng/ml males; 1.3 ± 0.21 ng/ml females). Experimental Series 2: Corticotropin-Releasing Hormone Injections Sea lamprey injected with 100 µg/kg of lamprey-CRH showed significantly (P<0.001) increased plasma concentrations of S after 1 h (7.4 ± 1.2 ng/ml males; 3.8 ± 0.29 ng/ml 30  females) compared to fish injected with saline only (1.4 ± 0.12 ng/ml males; 1.4 ± 0.09 ng/ml) (Fig 2.2). Levels of S remained significantly elevated (P<0.01, P<0.001) 24 h after injection (7.9 ± 0.97 ng/ml males; 3.1 ± 0.52 ng/ml females) compared to controls at 24 h (3.2 ± 1.7 ng/ml males; 1.3 ± 0.09 ng/ml females). Further characterization of this response indicated a dose-dependent increase of S in Pacific lamprey (Fig 2.3). Males exhibited a significant (P<0.01, P<0.001) response to CRH injected intraperitoneally at a dose of 50 µg/kg (6.5 ± 0.82 ng/ml) and 100 µg/kg (6.5 ± 0.82 ng/ml) compared to groups injected with saline only (1.2 ± 0.14 ng/ml). There was a trend toward an increase of S at 0.1 µg/kg, although this was not statistically significant. A qualitatively similar response was observed in females, where injection of 0.1, 50, and 100 µg/kg of CRH significantly (P<0.01, P<0.001) increased plasma levels of S to 3.5 ± 0.81 ng/ml, 3.7 ± 0.31 ng/ml, and 5.2 ± 0.50 ng/ml respectively, compared to 1.2 ± 0.08 ng/ml for the saline injected animals. Confirmation of 11-Deoxycortisol Confirmation of the identification of S in the plasma of male and female sea lamprey and Pacific lamprey from the Experimental Series 2: CRH injection experiments was obtained by fractionating steroid extracts from plasma samples of sea lamprey and Pacific lamprey that had been injected with 100 µg/kg of CRH by HPLC and then performing RIA. For all samples, RIA of the HPLC-separated product indicated only single immunoreactive peaks in the fractions that co-eluted with standard S (Fig 2.4).  31  Experimental Series 3: Adrenocorticotropic Hormone Injections The overall effect of treatment with ACTH varied by isoform as well as by sex (Fig 2.5 A / B); in some cases levels of S tended to increase in response to dose, although this effect was not significant. Male sea lamprey injected with 10 µg/kg of ACTH1–59, ACTH1– 59;35P,  and ACTH1–60;35P, produced statistically significant (P<0.05, P<0.001) levels of S (1.52  ± 0.14 ng/ml, 1.49 ± 0.14 ng/ml, and 1.99 ± 0.20 ng/ml, respectively), compared to animals injected with saline only (0.80 ± 0.04 ng/ml) (Fig 2.4 A). The mean concentration of S for the positive control (100 µg/kg of CRH) was 1.73 ± 0.24 ng/ml (P<0.001). No significant response to any isotope was observed at a dose of 1 µg/kg. In female sea lamprey, significant (P<0.05) increases in plasma S were observed in response to injection of 10 µg/kg of ACTH1–59; 35P, ACTH1–60, and ACTH1–60;35P, (1.55 ± 0.22 ng/ml, 1.51 ± 0.41 ng/ml, and 1.49 ± 0.18 ng/ml, respectively) (Fig 2.4 B). The only isoform that elicited a significant response at 1.0 µg/kg was ACTH1–60; 35P (1.38 ± 0.20 ng/ml). The mean concentration of S for the positive control (100 µg/kg of CRH) was 1.64 ± 0.16 ng/ml (P<0.01). Experimental Series 4: In Vitro Corticosteroidogenesis In vitro incubations of male and female sea lamprey tissues indicate that only the mesonephric kidney is able to produce significant (P<0.05, P<0.001) levels of S (Fig 2.6 A / B). For males, mean concentration of S per mg of tissue incubated was 1.1 ± 0.52 pg/mg in the absence of ACTH and 1.3 ± 0.38 pg/mg in the presence of ACTH, with no significant difference between the two groups (Fig 2.6 A). For female fish, mean concentration of S per  32  mg of tissue incubated was 4.5 ± 1.7 pg/mg in the absence of ACTH and 2.3 ± 0.17 pg/mg in the presence of ACTH, with no significant difference between the two groups (Fig 2.6 B). For both sexes, production by the gonads, gill, and liver was negligible, although in the case of the gonads not below the assay detection limit prior to controlling for mass. Incubations with tritiated precursor steroids indicated that only 17αP was biotransformed into S (Fig 2.7 – 2.9 A/B). Preliminary identification of S was obtained by running incubation media on TLC; co-migration with standard S occurred only in samples incubated with tritiated 17αP (Fig 2.7 A/B). Confirmation of the identification of the putative S was obtained by fractionating incubation media of samples containing 17αP by HPLC (Fig 2.10 A-B), which yielded up to four peaks. In all cases, peaks corresponding to the elution point of standard S were observed, with a mean rate of conversion from 17αP to S of 17 ± 3.8 % in males and females. Further identification was obtained by running the HPLC-purified fractions that coeluted with standard S on TLC both before and after acetylation. In both males (Fig 2.11 A/B) and females (Fig 2.12 A/B) results of radioactive co-migrations show peaks corresponding exclusively to standard S and acetylated-standard S.  Discussion Results from each of the Experimental Series provide support for the hypotheses that lamprey have a functional HPI stress axis that is similar to that of more derived species. Specifically, plasma concentrations of 11-deoxycortisol increased 1 h following exposure to acute stress by air-exposure, after intraperitoneal injection of lamprey-CRH, and after 33  intraperitoneal injection of lamprey-ACTH. Lamprey mesonephric kidneys produced significant amounts of S when incubated in L-15 medium only, and also biotransformed tritiated 17α-hydroxyprogesterone to S. These findings are consistent with the recent data published on lamprey, and provide evidence that lamprey have a functional HPI axis. Basal Stress Levels Wide variations exist in the endocrine stress response in fish, both between species of the same genus, as well as between wider taxonomic groups (Barton and Iwama, 1991; Gamperl et al., 1994; Mommsen et al., 1999; Barton, 2002). A trend has emerged showing that more recently derived species such as salmon and trout elevate plasma cortisol concentrations up to two orders of magnitude more than more basal vertebrates such as sturgeon and paddlefish (Barton, 2002). Other basal vertebrates such as elasmobranchs also show relatively low levels of their primary stress hormone, 1α-hydroxycorticosterone (Manire et al., 2007; Anderson 2011), and the oldest extant vertebrates, lampreys, exhibit low levels of 11-deoxycortisol that also fit with this trend (Close et al., 2010). In the present study, levels of S in control groups were found to be 1.1 ± 0.3 ng/ml (mean ± SE) in sea lamprey and 1.4 ± 0.2 ng/ml in Pacific lamprey. These match levels previously measured in lamprey species by Close et al., (2010), and are on the low end of the range compared to cortisol levels reported for fish in general. Basal levels of cortisol in fish have been reported over the range of 1 - 75 ng/ml (Sumpter et al., 1986; Pickering and Pottinger 1985; Pottinger and Moran, 1993; Gamperl 1994 ; Di Marco et al., 1999; Barton, 2002; Ceracato et al., 2009). Concentrations have been found to vary widely between species, and in certain cases intraspecies variation is also markedly high: basal levels have 34  been reported between 1 – 10 ng/ml in trout (Sumpter et al., 1986; Pickering and Pottinger 1985; Pottinger and Moran, 1993), between 2 – 13 ng/ml in sturgeon (Di Marco et al., 1999; Barton, 2002), 2 – 48 ng/ml in Atlantic salmon (Pickering and Pottinger 1983; Einarsdottir et al., 1996), and 50 – 75 ng/ml in catfish (Strange 1980; Barcellos 2001; Cericato 2009). Variations in reported plasma cortisol concentrations may reflect a number of factors including differences in sampling techniques, previous exposure to stressors, fish age, size, life stage, season, collection habitats, and rearing temperature (Pickering and Pottinger 1983; Sower et al. 1985; Feist et al. 1990; Cataldi et al., 1998; Di Marco et al., 1999). Furthermore, a number of explanations have been put forward regarding the timing and extent of cortisol responses including genetic, developmental, and environmental factors, which combine to play a role in the promotion of acclimation and survival by the responses of neural, cardiovascular, autonomic, immune and metabolic systems (Barton, 2002; McEwan 2008). Experimental Series 1: Acute Stress Treatment Following exposure to acute stress, concentrations of plasma cortisol in fish have been found to range between 30 – 310 ng/ml, and generally peak between 30 – 60 min, depending on the type, duration, and severity of the acute stressor (Wedemeyer et al., 1990; Barton and Iwama, 1991; Gamperl et al., 1994). The rapidity of the cortisol response also varies greatly. Several species including Atlantic salmon (Einarsdottir and Nilssen, 1996), tilapia (Foo and Lam, 1993; Pepels et al., 2004), perch (Carragher and Rees, 1994), coral reef fish (Grutter and Pankhurst, 2000), and catfish (Barcellos et al., 2001) have been shown to exhibit an extremely rapid cortisol response, with significant elevation occurring 35  by 5 minutes, a maximum response by 30 minutes, and a return to near pre-stress levels within 1 - 2 h. These species also tend to be more recently derived, and have higher basal cortisol levels, higher peaks, and wider intraspecies variation. Other, more basal species tend to exhibit longer times to peak responses: between 30 - 60 min for sturgeon (Belanger et al., 2001), 30 - 60 min for lamprey (Close et al., 2010), up to 4 h for sea raven (Vijayan and Moon, 1994), and potentially even longer in sharks (Manire et al., 2007). Following exposure to acute stress, lamprey in this study exhibited a response consistent with that found by Close et al., (2010) as well as with other fish in general (Barton and Iwama, 1991; Barton, 2002). This current study is the first to measure acute stress in both male and female adult Pacific lamprey. Results show a peak of 6.7 ± 1.2 ng/ml S for males and 4.3 ± 1.0 ng/ml S for females at 1 h post-stress, two- to three-times higher than the levels found by Close et al. (2010). Although the difference between the two studies is large, the different species used and the life stages of the animals may account for the discrepancy. Plasma S returned to pre-stress levels by 8 h. No production of S was measured before 10 min. This study demonstrates that the response to acute stress in lamprey is similar to that of other fish, and is consistent with the observed trend that more basal species have slower response times and lower maximum levels of stress hormones. This provides strong supporting evidence that S is indeed the corticosteroid naturally produced by lamprey in response to stress, and that a measured increase in its plasma concentration is indicative of physiological stress.  36  Experimental Series 2: Corticotropin-Releasing Hormone Injections Adult sea lamprey injected intraperitoneally with 100 µg/kg of lamprey-CRH showed significantly higher levels of 11-deoxycortisol production at 1 h post-injection compared to animals injected with saline only. These values were more than double those found by Close et al., (2010) in parasitic (non-mature) lamprey after injection of 100 µg/kg of human-CRH. No other comparable studies exist measuring S in lamprey. Possible explanations for these differences include the different species and life stages of the animals. A second explanation may be that the peptide based on sea lamprey sequences is more effective at stimulating the stress response than one based on human sequences. Regardless, the stimulation of S production by sea lamprey-CRH confirms that this peptide is functional in lamprey. Once the biological activity of the lamprey-CRH peptide was validated, the following experiments were performed on Pacific lamprey due to availability during adult life stages. Pacific lampreys injected intraperitoneally with CRH (0.1, 50, or 100 µg/kg) or saline solution (0.90% NaCl) showed production of S that was sensitive to dose. Although the dose of 100 µg/kg was kept consistent with the previous experiment, the lower limit of CRH efficacy in lamprey was unknown. Most studies performing CRH injections in other fish have tended to use much higher doses of the peptide (Clements et al., 2002; Clements and Schreck, 2004; Wang et al., 2004). However, the lower limits reported are in the range of 0.1 - 1 µg/kg for fish (Baker et al., 1996; Rotllant et al., 2001), and between 0.03 µg/kg and 0.1 µg/kg for mammals (Raisanen et al., 1990; Lavicky et al., 1993; Gupta et al., 2004). The lower limit was accordingly set at 0.1 µg/kg. Because it was uncertain whether this 37  dose, or indeed whether 10x or 100x this dose, would stimulate significant S production, and due to the limited number of fish available for testing, the mid-level dose was set at 50 µg/kg. This was also in keeping with Wang et al., (2004) who demonstrated that 50 µg/kg stimulated significant levels of cortisol in bass. Following the injections, a dose-depended response was observed in both males and females. Levels of S in control groups were consistent with those measured in sea lamprey, and showed very little variation. Results from Experimental Series 2: CRH injection experiments support my second hypothesis, demonstrating that production of S in lamprey is sensitive to dose of CRH in the same way as cortisol is in teleost fish. Confirmation of 11-Deoxycortisol In species that have not been as thoroughly studied as popular model organisms or those with large economic importance, there are difficulties associated with screening or detection based solely on the use of antibodies (Markov, 2008). It may be difficult to determine whether the reagents are able to recognize only the steroid in question, or whether they also show cross-reactivity with other closely-related molecules. Early studies on corticosteroids in lamprey repeatedly reported the presence of cortisol (Phillips et al., 1962; Buus and Larsen, 1975; Weisbart et al., 1980; Adams et al., 1987), whose presence was later refuted (Close et al., 2001; Close et al., 2010), indicating that relying on a single method of identification is likely to provide false-positives. In order to confirm the identity of the corticosteroid being measured by RIA in the Experimental Series 2: CRH-injections, extracts of plasma samples from both sea lamprey  38  and Pacific lamprey were fractionated by HPLC and re-assayed with S antibody as previously described by Close et al., (2010). The results show a single immunoreactive peak at the fraction that co-elutes with standard S, indicating that there was no crossreactivity between the antibody and any other steroids present in plasma extract. Experimental Series 3: Adrenocorticotropic Hormone Injections Sea lamprey were injected with ACTH at either 0.1 µg/kg or 10 µg/kg, or saline solution (0.90% NaCl), doses that agree with amounts used in earlier reports. Although several studies have used doses of 100 µg/kg and much higher in both fish and mammals (Oelkers et al., 1988; O’toole et al., 1990; Belanger et al., 2001; Cericato et al., 2009), the minimum effective dose of ACTH for stimulation of cortisol through the HPI/A axis appears to be between 0.1 – 10 µg/kg in both classes (Rock et al., 1984; Thomas and Robertson, 1991; Dickstein et al., 1997; Belanger et al., 2001; Madej et al., 2005). ACTH also stimulates 1α-hydroxycorticosterone in sharks, although the extent to which it does so has been difficult to quantify due to difficulties developing effective assays for this modified corticosteroid (Norris and Carr, 2006; Manire et al., 2007). It also remains unknown which sequences of the peptide are necessary to stimulate biological activity; mammalian ACTH requires only the first 24 amino acids including the NH2 terminal (Li, 1963; Evans et al., 1966; Hadley, 1992). Due to post-translational modifications, lamprey have four unique isoforms of ACTH, whereas all other vertebrates have one (Kawauchi and Sower, 2006; Norris, 2007). Additionally, lamprey ACTH peptides are 20-21 amino acids longer than those of other vertebrates, and show very little similarity to more derived species (for in-  39  depth discussions, see Takahashi and Kawauchi, 2006; Kawauchi and Sower, 2006; and Takahashi et al., 2006). Certain isoforms of ACTH significantly increased plasma concentrations of S over control levels in both males and females. However, the specific peptides and the levels to which they were effective differed between the sexes. Although 10 µg of the nonphosphorylated ACTH1–59 stimulated production of S in males, and 10 µg of the nonphosphorylated ACTH1–60 did so in females, only the phosphorylated ACTH1–59;35P and ACTH1–60;35P peptides stimulated production in both sexes. Furthermore, only ACTH1–60; 35P showed any effectiveness at 1.0 µg/kg. This may indicate that the phosphorylation at position 35 resulting from post-translational modification may have a role in the binding specificity to the receptors, although further studies will be required to determine whether the phosphorylation is in fact required to make a significant difference. Despite the fact that the overall response to stress appeared to be depressed in the entire collection of lamprey, significant differences between treatment and control groups were found. Furthermore, the positive control (CRH) group did show significantly elevated plasma 11-deoxycortisol concentrations, indicating that the response to stress was indeed functional and responsive. These data provide supporting evidence for my third hypothesis, demonstrating that injection of ACTH stimulates production of S in the same way as it does cortisol in teleost fishes.  40  Experimental Series 4: In Vitro Corticosteroidogenesis This is the first study to provide direct and conclusive evidence that the mesonephric kidneys of lamprey are able to produce S. The first evidence for this was obtained through RIA analysis of incubation media. Of the four tissues incubated in vitro, only the kidneys produced a significant amount of immunoreactive steroid. Tissues were also incubated in the presence of 100 ng/ml each of four isoforms of ACTH in order to determine whether their presence would affect production of S. It has previously been demonstrated that ACTH at doses between 1 ng/ml and 1000 ng/ml stimulates in vitro cortisol production in the interrenal cells of a number of species including salmon (Young, 1988), trout (Rance and Baker, 1981; Barry et al., 1995), sea bream (Rotllant et al., 2000), carp (Metz et al., 2005), and the adrenal cortex of mammals (Peron and Koritz, 1958; Schimmer, 1995). The dose of ACTH added in the incubations of the present study (100 ng/ml) matches the range of those previously used. The presence of ACTH, however, had no significant effect on the level of in vitro production of S. One possible explanation is that a secondary messenger such as cAMP is required. For example, in vitro production of cortisol is dependent upon the presence of both ACTH as well cAMP in tilapia (Balm et al., 1994; Quabius et al., 1997), trout and salmon in certain life stages (Brodeur et al., 1998; McQuillan et al., 2003), as well as a number of mammals (Schimmer, 1995). A second factor may be that the fish in this study were near sexual maturation, a factor that is known to affect in vivo sensitivity to circulating ACTH levels and production of cortisol and corticosteroids in general (McQuillan et al., 2003). It may be that a higher dose of ACTH is needed to effectively 41  stimulate production of S. Thirdly, this study included all four isoform of ACTH together, which may have caused interference in the signalling or production. Once future studies determine which isoforms are most effective at stimulating S in vivo, and whether a secondary messenger such as cAMP is required, this question can be revisited. The next piece of supporting evidence that it is S being produced by the kidney was made after in vitro incubations involving the addition of tritiated precursor steroids. Preliminary identification of S was made by running incubation media on thin layer chromatography (TLC) and noting co-migration with standard S. Peaks of radioactivity corresponding to the migration position of standard S occurred only in media to which 17αP was added, indicating the presence of a 21α-hydroxylase in the kidney. It is unknown whether the pathway of biotransformation occurs in the same order as in teleost fishes, or whether secondary messengers such as cAMP are required for activation of 3βhydroxysteroid dehydrogenase or 21α-hydroxylase. It has been demonstrated that tilapia, salmon, and trout produce cortisol by way of pregnenolone, 17α-pregnenolone, 17αprogesterone, 11-deoxycortisol, and finally cortisol, although other accounts indicate that the hydroxylations at positions C-17, C-21, and C-11 may occur in any order (Arai et al., 1969; Sangalang et al., 1972; Balm et al., 1989). Further identification of S was made by analyzing DPM counts of samples fractionated by HPLC. Radioactive peaks corresponding to the elution point of standard S occurred in both male and female samples. Several other peaks appeared, indicating the production of unidentified metabolic products. It also appears that the presence of ACTH increased conversion of 17αP to S in females, but not in males. Previous studies analyzing 42  the biotransformation of steroids have shown transformation of 17αP to cortisol in the interrenal cells of several teleosts, with conversion rates between 17 and 30% (Arai et al., 1969; Colombo et al., 1972; Sangalang et al., 1972; Balm et al., 1989). The conversion rate in the present study of 17% matches these previous accounts. The final confirmation of the identity of the presumptive S was made after noting co-migration on TLC of HPLC-purified product with standard S both before and after acetylation. This method of acetylation has been used for nearly half a century to accurately identify steroids produced by several tissues in vitro in a number of species (Berliner, 1957; Dominguez, 1963; Kawagishi et al., 1988). Furthermore, Bryan et al., (2003; 2004) have recently used this technique to differentiate between steroids and very structurally similar metabolic products in lamprey. Taken together, these in vitro incubation experiments provide very strong evidence for my fourth hypothesis that S is produced by the kidney in lampreys: (1) the product shows immunoreactivity with S-antibody, (2) it co-migrates with standard S on TLC, (3) it co-elutes with standard S on HPLC, and (4) HPLC-purified product co-migrates with standard S on TLC before and after acetylation.  43  Plasma 11-deoxycortisol (ng/ml)  10.0 Female Male  * * * ** *  8.0  6.0  4.0  2.0  0.0 0  5  10  60  480  1440  Time (min) Figure 2.1. Plasma 11-deoxycortisol concentration of adult male (n =3-5) and female (n = 4-7) Pacific lamprey after acute stress by air-exposure. Data are mean ± SE. Asterisks indicate a significant (*P < 0.05; **P < 0.01; ***P < 0.001) difference relative to controls.  44  Plasma 11-deoxycortisol (ng/ml)  10.0  * * *  Male Saline Male CRH Female Saline Female CRH  *  8.0  6.0  * ** *  4.0  *  * *  2.0  0.0 1  24  Time (hr) Figure 2.2. Plasma 11-deoxycortisol concentration of adult male (n =6) and female (n =6) sea lamprey after intraperitoneal injection of lamprey corticotropin-releasing hormone (100 μg/kg body weight) or saline solution (0.90% NaCl). Data are mean ± SE. Asterisks indicate a significant (*P < 0.05; **P < 0.01; ***P < 0.001) difference relative to controls.  45  Plasma 11-deoxycortisol (ng/ml)  8.0  * * *  Female Male  6.0  * * ** 4.0  * * *  * *  2.0  0.0 0  0.1  50  100  CRH (g/kg) Figure 2.3. Plasma 11-deoxycortisol concentration of adult male (n =5) and female (n = 5) Pacific lamprey after intraperitoneal injection of lamprey CRH or saline solution (0.90% NaCl). Data are mean ± SE. Asterisks indicate a significant (*P < 0.05; **P < 0.01; ***P < 0.001) difference relative to controls.  46  S  B Plasma 11-Deoxycortisol (ng/ml)  Plasma 11-Deoxycortisol (ng/ml)  A 6.0  5.0  4.0  3.0  2.0  1.0  0.0  6.0  S  5.0  4.0  3.0  2.0  1.0  0.0 40  41  42  43  44  45  46  47  48  49  50  40  41  42  43  HPLC fraction No.  45  46  47  48  49  50  HPLC fraction No.  S  D  7.0  Plasma 11-Deoxycortisol (ng/ml)  Plasma 11-Deoxycortisol (ng/ml)  C  44  6.0  5.0  4.0  3.0  2.0  1.0  0.0  6.0  S 5.0  4.0  3.0  2.0  1.0  0.0  40  41  42  43  44  45  46  HPLC fraction No.  47  48  49  50  40  41  42  43  44  45  46  47  48  HPLC fraction No.  Figure 2.4. Plasma 11-deoxycortisol concentration from adult (A) male sea lamprey, (B) female sea lamprey, (C) male Pacific lamprey, and (D) female Pacific lamprey following fractionation by HPLC. Arrow shows the elution point of standard S.  47  49  50  A (  B (  Figure 2.5. Plasma 11-deoxycortisol concentration of (A) adult male (n =5), and (B) female sea lamprey after injection with four isoforms of lamprey adrenocorticotropic-hormone or saline solution (0.90% NaCl) (59, 59P, 60, and 60P refer to the peptide length and phosphorylation state; C denotes saline-injected animals). Data are mean ± SE. Asterisks indicate a significant (*P < 0.05; **P < 0.01; ***P < 0.001) difference relative to controls. 48  A  11-deoxycortisol (pg/mg)  7.0 Male control Male ACTH  6.0  5.0  4.0  3.0  *  2.0  *  1.0  0.0  Kidney  Gonad  Gill  Liver  B 7.0  11-deoxycortisol (pg/mg)  *  Female control Female ACTH  6.0  5.0  4.0  3.0  * **  2.0  1.0  0.0  Kidney  Gonad  Gill  Liver  Figure 2.6. Concentration of 11-deoxycortisol in tissues of (A) adult male (n=3) and (B) female sea lamprey after incubation in the presence or absence of four isoforms of lamprey ACTH. Data are mean ± SE. Asterisks indicate a significant (*P < 0.05; **P < 0.01; ***P < 0.001) difference relative to controls.  49  A 5000  S  Male control Male ACTH  4000  3H DPM  Preg 3000  2000  1000  0 0  5  10  15  20  TLC Section No.  B 5000  S  Female control Female ACTH  4000  3H DPM  Preg 3000  2000  1000  0 0  5  10  15  20  TLC Section No.  Figure 2.7. 3H counts (DPM) from TLC fractionation of incubation media from (A) male and (B) female sea lamprey mesonephric kidneys incubated with tritiated pregnenolone in the presence or absence of ACTH. Arrows show the migration points of standard 11deoxycortisol and pregnenolone.  50  A 5000  S  Male control Male ACTH  3H DPM  4000  P  3000  2000  1000  0 0  5  10  15  20  TLC Section No.  B 5000  S  Female control Female ACTH  4000  3H DPM  P 3000  2000  1000  0 0  5  10  15  20  TLC Section No.  Figure 2.8. 3H counts (DPM) from TLC fractionation of incubation media (A) male and (B) female sea lamprey mesonephric kidneys incubated with tritiated progesterone in the presence or absence of ACTH. Arrows show the migration points of standard 11deoxycortisol and progesterone.  51  A 5000  S Male control Male ACTH  3H DPM  4000  17P  3000  2000  1000  0 0  5  10  15  20  TLC Section No.  B 5000  S  Female control Female ACTH  3H DPM  4000  17P  3000  2000  1000  0 0  5  10  15  20  TLC Section No.  Figure 2.9. 3H counts (DPM) from TLC fractionation of incubation media (A) male and (B) female sea lamprey mesonephric kidneys incubated with tritiated 17αOH-progesterone in the presence or absence of ACTH. Arrows show the migration points of standard 11deoxycortisol and 17αOH-progesterone.  52  A  S  17P  40000  Male control Male ACTH  35000  3H (DPM)  30000 25000 20000 15000 10000 5000 0 20  30  40  50  60  70  80  HPLC fraction no. S  B  17P Female control Female ACTH  160000 140000  3H (DPM)  120000 100000 80000 60000 40000 20000 0 20  30  40  50  60  70  80  HPLC fraction no.  Figure 2.10. 3H counts (DPM) of 17αP incubation media from (A) male and (B) female sea lamprey fractionated by 90 min HPLC runs. Arrows show the elution points of standard S and 17αP.  53  A 20000  S S-Acet  S-Acet  S  3 H DPM  15000  10000  5000  0 0  5  10  15  20  TLC Section No.  B S S-Acet  20000  S  S-Acet  3H DPM  15000  10000  5000  0 0  5  10  15  20  TLC Section No.  Figure 2.11. 3H counts (DPM) from TLC fractionation of male sea lamprey (A) control and (B) ACTH incubation media after purification with HPLC. Black bars represent the purified product of incubation, and grey bars represent the acetylated product. Arrows show the migration points of standard S and acetylated S.  54  A 20000  S-Acet  S  S S-Acet  3H DPM  15000  10000  5000  0 0  5  10  15  20  TLC Section No.  B  S-Acet  S  20000  S S-Acet  3H DPM  15000  10000  5000  0 0  5  10  15  20  TLC Section No.  Figure 2.12. 3H counts (DPM) from TLC fractionation of female sea lamprey (A) control and (B) ACTH incubation media after purification with HPLC. Black bars represent the purified product of incubation, and grey bars represent the acetylated product. Arrows show the migration points of standard S and acetylated S.  55  Chapter 3: Conclusion Summary The data presented in this thesis provide four lines of evidence to support the hypothesis that lamprey have a functional HPI stress axis that similar to that of teleost fishes. First, in response to acute stress by air-exposure, plasma concentrations of 11deoxycortisol were found to increase after 1 h in Pacific lamprey. This matches the response time of other fish, and also fits with the observed trend that more basal species have a slower initiation of the stress response than more derived species. Levels of S were higher than those previously reported, although the difference in species used and life stages may likely account for the discrepancy. Second, intraperitoneal injection of CRH manufactured based on sequences from the sea lamprey genomic database increased plasma concentrations of S in sea lamprey, and in a dose-dependent manner in Pacific lamprey that matches observations for cortisol in teleosts. CRH has a highly conserved structure, and the results support previous observations that it has a highly conserved function. Third, intraperitoneal injection of lamprey-ACTH based on the sequences described by Takahashi et al. (2006) increased plasma S concentrations in Pacific lamprey. Although a dose-dependent response was not observed, a statistically-significant effect was found. ACTH has a much more variable structure across species than CRH, and only the first 24 amino acids of 40 are required for stimulation of cortisol in teleosts. The large structural difference but conserved function may indicate an ancestral function of the peptide. 56  Fourth, this study demonstrates that the mesonephric kidney of sea lamprey can produce S in vitro in the presence of incubation media only, and also from the biotransformation of the precursor steroid 17αP. Previous studies have suggested the presence of a presumptive adrenocortical tissue located behind the heart (Weisbart and Youson, 1975), but have not used the multiple levels of confirmation presented here. Overall, the three stages of the HPI axis resulting from sensitivity to CRH, ACTH, and site of production in the kidney describe a response to stress that is similar to that of more derived fishes.  Thesis Objectives and Hypotheses Regarding the initial objective of this thesis: this study provides strong supporting evidence for the hypothesis that lamprey have a functional HPI axis and that their stress response functions in a manner similar to that of teleost fishes. Specifically: Objective 1: Plasma 11-deoxycortisol concentrations increase 1 h following acute stress by air-exposure. Hypothesis 1: The results of these experiments support the hypothesis that plasma 11deoxycortisol concentrations would increase 1 h following acute stress. Objective 2: Lamprey-CRH stimulated production of 11-deoxycortisol in both sea lamprey and Pacific lamprey. This production was sensitive to dose. Hypothesis 2: The results of these experiments support the hypothesis that intraperitoneal injection of lamprey-CRH would increase plasma concentrations of 11-deoxycortisol 1 h following injection. 57  Objective 3: Certain isoforms of lamprey-ACTH stimulated production of 11-deoxycortisol in sea lamprey. This production may be sensitive to dose, although the extent to which this occurs is unclear as the overall stress response in the experimental animals appears to have been depressed. Hypothesis 3: The results of these experiments support the hypothesis that intraperitoneal injection of lamprey-ACTH would increase plasma concentrations of 11-deoxycortisol 1 h following injection. Objective 4: 11-deoxycortisol is produced in the mesonephric kidney both in the presence and absence of the precursor steroid 17α-hydroxyprogesterone. Hypothesis 4: The results of these experiments support the hypothesis that 11-deoxycortisol would be produced by the mesonephric kidney.  Implications Research into the physiological mechanisms of animals’ response to stress has become increasingly important and applicable to a variety of real-world issues. One example is the recent realization that the tissues and molecules involved in the stress response often have secondary or interrelated roles with the immune or neuroendocrine systems. Furthering our understanding about the stress response in a variety of vertebrate species can help elucidate the interactions between body systems and the impacts that environmental change, illness, or medication may combine to have. These effects also have economic consequences, as the success of agriculture and aquaculture operations depends primarily on the health and growth rate of the animals. When animals in commercial operations are exposed to acute stress, such as overcrowding, rough handling, and 58  transport, they show decreased growth rates. If the stressors persist chronically, mortality rates rise. Increasing our understanding of the primary stages of the stress response can lead to better management for animal husbandry and health, and thus to better economic earnings. Studying a basal vertebrate such as the lamprey can also shed light on how the stress system of different classes of vertebrates evolved to their current states. Differences and similarities between fish and mammals for example can be more clearly understood if an ancestral state from which they both evolved can be described. Although the exact phylogenetic relationships between early vertebrates and jawed vertebrates remain a matter of debate, especially as different evolutionary models arise from different lines of evidence, common ideas and trends will emerge that will allow definite interpretations in comparative physiology (Smith et al., 2010).  Limitations The results of these experiments provide new insights that help fill major gaps in our current understanding of the early vertebrate stress response. However, there are three major interrelated and limiting factors that should be taken into consideration during the discussion and interpretation of the results. First to be considered is the fact that these are some of the first experiments of their kind to have ever been performed on lamprey. Compared to the progress and rate of studies carried out in other species such as salmon and other economically-valuable fish, research into the stress response of lamprey remained essentially at a standstill from the early 1980s until the recent breakthrough of Close et al., in 2010. Accordingly, while certain 59  similarities between lamprey and more derived species are described, it is important to recognize that the discoveries presented here are completely novel, and the ideas they address were a matter of controversy and conflicting results over the past half-century. The second consideration expands on the fact that these data are the first of their kind in any agnathan, and this study should be seen as a starting point for future, more directed studies, rather than a way to immediately begin drawing evolutionary- and physiologically-based conclusions. This is an important consideration that deserves explicit mention because in the past decade, certain studies have taken new or sensational data from studies in lamprey to draw very specific conclusions about the evolutionary origins of certain physiological mechanisms without the support provided by subsequent or repeated studies. Thirdly, lampreys belong to a very phylogenetically isolated genus; the closest living relatives of lamprey are hagfish, animals whose stress response is even more poorly understood – sliming novelties aside. There are no easily comparable species, and accordingly, comparisons of the results presented in this thesis have been made to a variety of fish species ranging from basal to more derived. It is thus up to future studies to expand on the findings reported here and assess the use of such data for practical applications.  Future Directions There are several future experiments that could follow up on the research presented in this thesis to provide further clarifications of the effects observed and to provide further data on early vertebrate stress physiology. One future experiment could determine whether CRH increases in plasma after acute stress, and how the timing of this response compares to S production. Blood samples 60  could be collected at regular intervals between 0 min and 60 min to show increases in both CRH and S, and also at longer spaced intervals after 60 min to show a return to basal levels. A second experiment with CRH could use immunocytochemistry to stain CRH binding receptors in the hypothalamus. These two studies together would provide further evidence that the first step in the HPI axis is similar to that of teleosts: CRH is produced in the hypothalamus, and increases in concentration prior to increases in plasma concentrations of S. Repeat studies will also be needed to confirm the effects of the four ACTH peptides. While the production of S in response to ACTH was statistically significant, the entire stress response appeared to be depressed. New studies should be performed as close to the collection site as possible in order to minimize handling and transportation stresses. If active peptides are found, a second and third experiment showing ACTH increasing in plasma after acute stress, and ACTH-antibody binding in the pituitary would confirm the second step in the HPI axis similarities: ACTH is produced by the pituitary, and increases in concentration prior to S. Lastly, in vitro corticosteroidogenesis could be expanded. Incubations of presumptive adrenocortical tissue with complete purification steps could confirm whether it is able to produce S, or whether the product is a closely related metabolite. Incubations of the kidney could specifically isolate interrenal cells, and test whether the presence of ACTH or secondary messengers stimulates production S or biotransformation of precursor molecules.  61  Once these processes are better understood, studies examining the effects of acute and chronic stressors in natural environments could be performed in order to help population control and habitat management programs.  62  References Adams, M. A., Teeter, J. H., Katz, Y., Johnsen, P. B. (1987). Sex pheromones of the sea lamprey (Petromyzon marinus): Steroid studies. Journal of Chemical Ecology, 13 (2), 387395. Almeida, P. R., Silva, H. T., Quintella, B. (2000). 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