yin}. 5,3, .l- .0

a
u .‘ 1"
twin..’$uf“\“a.!

Bra. 1

u

Hﬂﬁ: .....r.m...mv3.-

_....,........f........r fawn} S.

umwsrta.

.21. him.

5...).

1...... ul.
1.9.! l.ﬂ...¢....,hl

éi.:c>7u-..Ml
znﬂwwf

 

 

" LIBRARY
2 008 Michigan State
University

 

 

This is to certify that the
dissertation entitled

CHARACTERIZATION OF AN ANCESTRAL GLUCOCORTICOID
HORMONE AND ITS COGNATE RECEPTOR IN THE SEA
LAMPREY

presented by
David A. Close

has been accepted towards fulﬁllment .
of the requirements for the .

PhD. degree in Fisheries and Wildlife

/
M/W‘V
Major Professor‘s Signature

/0 ”66944” 20757

Date

 

 

 

MSU is an aﬁ‘innative-action, aqua/opportunity employer

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DAIEDUE

DAIEDUE

DAIEDUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/07 p:/ClRC/DaleDue.indd-p.1

 

CHARACTERIZATION OF AN AN CESTRAL GLUCOCORTICOID HORMONE
AND ITS COGNATE RECEPTOR IN THE SEA LAMPREY

By

David A. Close

A DISSERTATION
Submitted to
Michigan State University
In partial fulﬁllment of the requirements
For the degree of
DOCTOR OF PHILOSOPHY
Department of Fisheries and Wildlife

2007

ABSTRACT

CHARACTERIZATION OF AN AN CESTRAL GLUCOCORTICOID HORMONE
AND ITS COGNATE RECEPTOR IN THE SEA LAMPREY

By

David A. Close

The goal of this research was to determine if one of the oldest extant vertebrates
possesses a stress steroid hormone. Previous studies have been unsuccessful in
determining if lampreys have a stress steroid. In order to conﬁrm that a molecule is a
hormone, the following criteria must be met: 1) deﬁnitive chemical identity, 2) a cognate
receptor for that molecule, and 3) the molecule must mediate biological effects.
Evidence is provided that 11-deoxycortisol is an ancestral stress steroid hormone in sea
lampreys (Petromyzon marinas). The current study demonstrated the isolation and
identiﬁcation of two putative glucocorticoids from the blood of the lamprey, 11-
deoxycortisol and ll-deoxycorticosterone. Both of these steroids were screened using
radioimmunoassays and isolated by chromatography techniques. Positive identiﬁcation
of both steroids was by use of mass spectrometry. The study provided evidence that 11-
Deoxycortisol has a receptor in the gill cytosol of the lamprey. The binding moiety for
ll-deoxycortisol showed high speciﬁcity, afﬁnity, and low capacity in the gill cytosol.
Speciﬁc binding was highest in the intestine followed by gill, testis, liver, kidney, heart,
and muscle. In addition, speciﬁc binding of the receptor complex to DNA was
demonstrated by DNA-cellulose chromatography. The study showed that circulating

levels of 1 l-deoxycortisol sharply increase after acute stress in spawning phase lampreys.

The stress hormone responds to acute stress in parasitic stage lampreys also. Slow
release ll-deoxycortisol implants increased circulating ll-deoxycortisol and decreased
classic androgens and estrogens. In addition, the implants increased gill Na+, K+-ATPase
activity in lamprey. The study demonstrated that mammalian corticotropin releasing
hormone increased ll-deoxycortisol. Pituitary extracts also increased circulating 11-
deoxycortisol in a dose-response manner. Overall, these results are the ﬁrst to
demonstrate that the ancestral vertebrate, the sea lamprey, has a stress steroid hormone.
In conclusion, this study demonstrated evidence for the identiﬁcation, receptor, and

biological effects of l l-deoxycortisol.

ACKNOWLEDGEMENTS

A special thanks to my advisor Dr. Weiming Li and the members of my graduate
committee, Dr. Tom Coon, Dr. Don Garling, and Dr. Ken Poff for their guidance and
support. A warm thanks to Dr. Sang-Seon Yun for guidance and support. Support from
Dr. Alexander Scott was appreciated. A special thanks to Dr. Brad Young for editing and
support. I am grateful to the staff of the US. Geological Survey, Hammond Bay
Biological Station for supplying laboratory space and sea lampreys. Bonneville Power
Administration for providing ﬁnding and support for this research. I am grateful to
Michigan State University and the National Institute of Health for fellowships. A warm
thanks to the Confederated Tribes of the Umatilla Indian Reservation for support of this

research.

iv

TABLE OF CONTENTS

LIST OF FIGURES .......................................................................................................... vii
INTRODUCTION ............................................................................................................... 1
Concept of stress ...................................................................................................... 1
Stress in ﬁsh ............................................................................................................. 4
Functions of corticosteroids ..................................................................................... 6
Lamprey as a model system ..................................................................................... 7
Introduction of dissertation ...................................................................................... 9
References .............................................................................................................. 11
CHAPTER 1
IDENTIFICATION OF PUTATIVE GLUCOCORTICOIDS IN THE SEA LAMPREY
Petromyzon marinas .......................................................................................................... 15
Abstract .................................................................................................................. 16
Introduction ............................................................................................................ 17
Materials and methods ............................................................................... 21
Maintenance of animals and blood collection ........................................... 21
Plasma extraction ....................................................................................... 21
High performance liquid chromatography ................................................. 22
Radioimmunoassay (RIA) and protein binding assay (PBA)
procedures .................................................................................................. 22
LH-20 column chromatography ................................................................. 23
Mass spectrometry analysis of plasma ....................................................... 23
Results .................................................................................................................... 25
Discussion .............................................................................................................. 32
Acknowledgements ................................................................................................ 36
References .............................................................................................................. 37
CHAPTER 2
CHARACTERIZATION OF A GLUCOCORTICOID RECEPTOR IN THE GILL OF
THE SEA LAMPREY Petromyzon marinas ..................................................................... 39
Abstract .................................................................................................................. 40
Introduction ............................................................................................................ 41
Materials and methods ............................................................................... 43
Animals and holding facilities ................................................................... 43
Materials .................................................................................................... 43
Preparation of cytosolic fractions of tissues .............................................. 43
Saturation curve and scatchard analysis .................................................... 44
Association and dissociation kinetics ........................................................ 45
Steroid and tissue binding speciﬁcity ........................................................ 45
DNA-cellulose chromatography ................................................................ 46
Results .................................................................................................................... 48

Discussion .............................................................................................................. 55

Acknowledgements ................................................................................................ 60
References .............................................................................................................. 61
CHAPTER 3
BIOLOGICAL ROLES OF ll-DEOXYCORTISOL IN MEDIATING STRESS
RESPONSES OF THE SEA LAMPREY Petromyzon marinas ....................................... 64
Abstract .................................................................................................................. 65
Introduction ............................................................................................................ 66
Materials and methods ............................................................................... 69
Source of animals ....................................................................................... 69
Handling and salinity stressors .................................................................. 69
Radioimmunoassay (RIA) procedure ........................................................ 7O
Steroid implants ......................................................................................... 71
Gill Na+, K+-ATPase activity ..................................................................... 71
Corticotropin releasing hormone injections ............................................... 72
Pituitary extract injections ......................................................................... 72
Statistical analysis ...................................................................................... 73
Results .................................................................................................................... 74
Discussion .............................................................................................................. 86
Acknowledgements ................................................................................................ 90
References .............................................................................................................. 91
SUMMARY OF DISSERTATION ................................................................................... 96

vi

LIST OF FIGURES

Chapter 1

Figure 1-1. Screening of HPLC fractions of lamprey plasma for cortisol-like steroids
using RIA and BPA. (A) Amount of immunoreactive cortisol found in 0.5 ml HPLC
fractions from RIA of 20 pl of each fraction and back calculated fraction volume. (B)
Amount of cortisol binding activity found in 0.5 m1 HPLC fractions from BPA of 20 ul
of each fraction and back calculated to fraction volume. An arrow shows where cortisol
and 11-deoxycortisol standard eluted. ir = immunoreactivity. .......................................... 27

Figure 1-2. Screening of HPLC fractions of lamprey plasma for corticosterone-like
steroids using RIA. Amount of immunoreactive corticosterone found in 0.5 ml HPLC
fractions from RIA of 20 ul of each ﬁ‘action and back calculated to fraction volume. An
arrow shows where corticosterone and 11-deoxycorticosterone elutes. ir =
immunoreactivity. .............................................................................................................. 28

Figure 1-3. Screening of LH-20 fractions of lamprey plasma for immunoreactive 11-
deoxycortisol and ll-deoxycorticosterone by RIA. Amount of immunoreactive 11-
deoxycortisol and ll-deoxycorticosterone found in LH-2O fractions from 800 ml of sea
lamprey plasma based on RIA of 20 pl of each fraction and back calculated to fraction
volume. ir, immunoreactivity ............................................................................................. 29

Figure 1-4. Identiﬁcation of 1 l-deoxycortisol in the plasma of sea lamprey. APCI
MS/MS analysis (positive mode) of authentic 11-deoxycortisol steroid from a 10 ug ml'1
stock solution (A) and HPLC fraction corresponding to immunoreactive fraction for 11-
deoxycortisol from 800 m1 lamprey plasma extract (B). ................................................... 30

Figure 1-5. Identiﬁcation of l 1-deoxycorticosterone in the plasma of sea lamprey. APCI
MS/MS analysis (positive mode) of authentic ll-deoxycorticosterone steroid from a 10

u g ml‘1 stock solution (A) and HPLC fraction corresponding to immunoreactive fraction
for 11-deoxycorticosterone ﬁ'om 800 ml lamprey plasma extract (B). ............................. 31

Chapter 2

Figure 2-1. Representative saturation curve (A) and hyperbolic regression plot (B) of 11-
dexoycortisol binding to gill cytosol. The abbreviations are; total binding (B7), speciﬁc
binding (B5), non-speciﬁc binding (BN5), maximum binding capacity of tissue (Bmax),

ﬁ'ee (i.e., unbound) [3 H] ll-deoxycortisol (F), and disintegrations per minute (DPM) are
shown; 11 = 3 ....................................................................................................................... 50

vii

Figure 2-2. Association and dissociation kinetics of a sea lamprey receptor in gill
cytosol. For association kinetics (A), T1 /2 was 2.11 d: 0.32 min, 11 = 3. For dissociation
kinetics (B), Tm was 26.44 :1: 8.41 min; n = 3. .................................................................. 51

Figure 2-3. Speciﬁcity of the lamprey gill cytosolic moiety for various steroids including
ll-deoxycortisol (S), ll-deoxycorticosterone (DOC), cortisol (F), corticosterone (B),
aldosterone (Aldo), Dexamethasone, (D), androstenedione (A), Estradiol (E2), 170(-
hydroxyprogesterone (17OHP), and progesterone (P); n = 3. ........................................... 52

Figure 2-4. Speciﬁc binding of [3H] ll-deoxycortisol (5 nM) to cytosolic moiety from
different tissues. Abbreviation; speciﬁc binding (B5). Vertical bars represent means :t
SEM; n = 3. ........................................................................................................................ 53

Figure 2-5. Elution of 1 l-deoxycortisol-receptor complex from DNA-cellulose. Gill
cytosol was preincubated with 20 nM [3 H] ll-deoxycortisol before binding to the column
of DNA-cellulose. Abbreviations are; total binding (BT), speciﬁc binding (BS),
nonspeciﬁc binding (BN3), and disintegrations per minute (DPM) are shown; 11 = 3 ........ 54

Chapter 3

Figure 3-1. Effect of handling and salt challenge stressors on plasma levels of 11—
deoxycortisol for spawning (A) and parasitic (B) phase lamprey during recovery.

Vertical bars represent means i SEM of 6-14 observations. *, P < 0.05; **, P < 0.01;
***, P < 0.001. ................................................................................................................... 78

Figure 3-2. Effect of 21 d ll-deoxycortisol implant treatment on plasma levels of 11-
deoxycortisol (A) and 11-deoxycorticosterone (B). Vertical bars represent means 3: SEM
of 1 1-12 observations. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ................................... 79

Figure 3-3. Effect of 21 d ll-deoxycortisol implant treatment on plasma levels of
Dehydroepiandrosterone-sulfate, DHEA-S (A) and Dehydroepiandrosterone, DHEA (B).
Vertical bars represent means 1: SEM of 11-12 observations. *, P < 0.05 ; **, P < 0.01;
***, P < 0.001. ................................................................................................................... 80

Figure 34 Effect of 21 d 11-deoxycortisol implant treatment on plasma levels of
androstenedione (A) and testosterone (B). Vertical bars represent means :1: SEM of 11-12
observations. *, P < 0.05; **, P < 0.01; ***, P < 0.001. .................................................. 81

Figure 3-5. Effect of 21 d ll-deoxycortisol implant treatment on plasma levels of estrone
(A) and estradiol (B). Vertical bars represent means i SEM of 11-12 observations. *, P
< 0.05; **, P < 0.01; ***, P < 0.001 .................................................................................. 82

Figure 3-6. Effect of 21 d ll-deoxycortisol implant treatment on gill Na+, K+-ATPase

activity. Vertical bars represent means i: SEM of 11-12 observations. *, P < 0.05; **, P
< 0.01; ***, P < 0.001. ...................................................................................................... 83

viii

Figure 3-7. Changes in concentration of plasma ll-deoxycortisol following injections of
mammalian CRH. Vertical bars represent means :1: SEM of 3-5 observations. *, P <
0.05; **,P<0.01; ***,P<0.001. .................................................................................... 84

Figure 38 Changes in concentration of plasma ll-deoxycortisol following injections of
pituitary extract. Vertical bars represent means :1: SEM of 9-10 observations. *, P < 0.05;
**,P<0.01; ***,P< 0.001. ............................................................................................. 85

ix

INTRODUCTION

Concept of stress

During the 19th century, physiologist Claude Bernard established the concept of a
constant “milieu interieur” or internal environment in the body (Cannon 1929; Robin
1979). Bemard’s concept in 1878 was that an organism regulates a stable internal
environment in an ever-changing external environment. Therefore, he is credited with
laying the foundation for the concept of homeostasis which was articulated during the
19205 (Cannon 1929). Cannon (1924) demonstrated that adrenal secretions were of
importance in maintaining homeostasis. Cannon (1929) actually coined the term
homeostasis and related it to maintaining an internal stable state. He also suggested that
there were two types of homeostasis involving supplies and processes to maintain a stable
state of the internal ﬂuid matrix. After Walter B. Cannon’s homeostasis concept came
the idea of a General Adaptation Syndrome by Hans Selye (1936).

Some of the most inﬂuential studies on stress response in animals were conducted
by Hans Selye. Selye (1936) described animals responding to stress in stages of alarm,
resistance and exhaustion. He called this phenomenon or theory the General Adaptation
Syndrome (GAS). The syndrome included morphological changes such as an
enlargement of the adrenal glands and atrophies of the thymus, spleen and lymphoid
tissues. If animals were exposed to continued stressors, they would develop resistance.
Eventually, the continuous stress would cause the animal to reach exhaustion and die. A

few years later, Selye (1946; 1950; 1956; 1973) was able to ﬁll in more details and

provided evidence that non-speciﬁc stressors elicited the alarm stage which activated the
pituitary-adrenal axis to release corticosteroids. Selye (1950) deﬁned the syndrome as
“the sum of all non-speciﬁc, systemic reactions of the body which ensue upon long
continued exposure to stress.” In addition, he divided the idea of stress into stressors and
stress response. Selye also reported that the adrenal glands were responsible for
“adaptation diseases” although this was later refuted (Sayers 1950). While some of the
details of Selye’s concept were proven incorrect through time (Munck et al. 1984), his
overall principle of the GAS concept was generally accepted.

During the 1980’s a different hypothesis was put forth regarding the role of the
adrenal stress steroids. Munck et al. (1984) hypothesized that the physiological function
of stress-induced increases in glucocorticoid levels is to protect not against the source of
the stress itself, but against the possible over-reaction of normal defense mechanisms
activated by stress. The glucocorticoids accomplish this function by inhibiting the
defense reactions, thus preventing them from overshooting and themselves threatening
homeostasis. Sapolsky et a1. (2000) provides a comprehensive review of glucocorticoid
actions associated with stress.

A relatively new concept of stress has emerged from the biomedical ﬁeld called
allostasis or allostatic load. Sterling and Eyer (1988) deﬁne allostasis as the process of
adaptation to acute stress including the ability to increase or decrease vital functions to a
new steady state on challenge. While this was a seemingly new terminology, others had
already explored the concept under different names. Schreck (1981) explored the
concept which he termed performance capacity in relation to stress compensation.

Several years later the concept was broadened to include energetic load (Schreck and Li

1991). McEwen and Stellar (1993) and McEwen (2000), concerned with diseases in
humans, coined the term allostatic load which was deﬁned as, “The strain on the body
produced by repeated upland downs of physiologic response, as well as by the elevated
activity of physiologic systems under challenge, and the changes in metabolism and the
impact of wear and tear on a number of organs and tissues, which can predispose the
organ to disease.” The primary purpose of the concept was to explore the linkage
between stress and disease at a ﬁner scale to uncover the mechanistic causes.

McEwen and Wingﬁeld (2003) expanded the allostasis load concept to include
Type 1 and Type 2 overload. The Type 1 overload means that energy demands exceed
the energy income, and what can be mobilized from stores. Type 2 occurs if energy
demands are not exceeded and the organism continues to take in or store as much or even
more energy than it needs. Therefore, Type 1 and Type 2 overloads are in addition to
normal allostatic load resulting from unpredictable events.

The role of glucocorticoids in mediating the effects of allostatic loading has been
reviewed by Landys et a1. (2006). After synthesis of the existing published data, they
found 3 levels of allostasis. The ﬁrst was a “low but critical basal level, the second an
elevated seasonal baseline that varies in conjunction with predictable demands, and the
third was a high stress-related level in response to unpredictable and life-threatening
perturbations.”

Fisheries biologists recognized the economic importance of the effects of stress in
ﬁsh hatcheries (Pickering 1981). Hatchery ﬁsh were regularly exposed to acute stressors

such as crowding, handling, and transporting. Sometimes the ﬁsh were exposed to

chronic stressors also which would lead to increased mortality. Therefore, it became

increasingly important for ﬁsheries biologists to quantify stress.

Stress in fish

Most ﬁsh biologists developed their ideas about stress from Hans Selye’s GAS
concept. Brett (1957) thought that Selye’s concept was too vague and ﬁsheries
professionals needed a better workable deﬁnition of stress. His deﬁnition is as follows:
“stress is a state produced by any environmental or other factor which extends the
adaptive responses of an animal beyond the normal range, or which disturbs the normal
functioning to such an extent that, in either case, the chances of survival are signiﬁcantly
reduced.” He thought that this deﬁnition would provide a more quantitative approach by
including estimates of chances of survival or by the measure of reduction in capacity of
performance. The concept of stress in ﬁsh was ﬁrrther expanded to include performance
capacity (Schreck 1981) and energetic load (Schreck and Li 1991) similar to the concepts
described earlier regarding allostasis. Pickering (1981) reviewed the evolution of the
stress concept as it relates to ﬁsh and provided accounts of ﬁsh biologists beginning to
quantify the stress response.

Instead of focusing on the concept of stress, ﬁsh physiologists focused efforts on
measuring and quantifying the stress response (Pickering 1981; Wedemeyer et al. 1990).
The new conceptual framework for stress in ﬁsh considered the stress response in terms
of primary, secondary, and tertiary changes (Mazeaud et al. 1977; Donaldson 1981;

Mazeaud and Mazeaud 1981; Wedemeyer and McLeay 1981). The primary response

was an endocrine system response which included the hypothalamus-pituitary—interrenal
axis (HPI). After a perceived stimulus by the central nervous system, catecholarnines and
stress steroids are released into circulation. Catecholarnines (for example, epinephrine),
which are the ﬁrst to increase in the blood, are released from the chromafﬁn tissues
located in various organs of different species (Mazeaud et al. 1977; Mazeaud and
Mazeaud 1981). Their principal role is thought to be the stimulation of glucose release
for immediate energy used in a ﬁght or ﬂight response. After the catecholamine response
is initiated, the stress steroid response is activated by the central nervous system. The
central nervous system stimulates Corticotropin Releasing Hormone (CRH) to be
released from the hypothalamus which in turn stimulates Corticotrophin Hormone
(ACTH) to be released from the pituitary. ACTH circulates and binds to receptors in the
interrenal cells of ﬁsh (homologue to adrenal gland) to stimulate the release of cortisol or
corticosterone (Donaldson 1981; Wendelaar Bonga 1997; Mommsen et al. 1999).

The secondary response includes changes in blood and tissue chemistry in
response to stress. Wedemeyer and McLeay (1981) and Wedemeyer et al. (1990)
describe these as secondary alterations in blood chemistry such as; hyperglycemia,
hyperlacticemia, hypochloremia, and reduced blood clotting time. In addition, they
describe measuring changes in liver glycogen or even reductions in vitamin C from
interrenal cells.

The tertiary response includes changes to individuals and populations
(Wedemeyer et al. 1990). Many of the tertiary responses were observed by hatchery
workers in daily operations (Wedemeyer and McLeay 1981). Some examples of tertiary

responses are reductions in growth rate, lowered resistance to disease, decreased

reproductive success, oxygen consumption, swimming performance, altered feeding
behavior or migratory behavior (Wedemeyer et al. 1990). Chronic stress may eventually

lead to reduced performance by individuals and thus eventually impact the population.

Functions of corticosteroids

The corticosteroids are a group of steroids produced and secreted by the adrenal
cortex cells in mammals or interrenal cells (in head kidney) in ﬁsh. In tetrapods, the
adrenal cortex is divided into three zones that produce three different classes of steroids.
The zones are the zona glomerulosa (minerlocorticoids), zona fasciculate
(glucocorticoids), and zona reticularis (androgens) (Norman and Litwack 1997).
Corticosteroids are divided into two subcategories: the glucocorticoids and
minerocorticoids. They are both characterized by a hydroxyl group at the 21 carbon
position. Further, glucocorticoids are characterized by the presence or absence of
hydroxyls at carbon 11 and carbon 17 positions. The principal glucocorticoids in
vertebrates are cortisol and corticosterone (Bern 1967; Idler and Truscott 1972; Sandor
1972; Jones et al. 1972) and are released in response to stressors. The only known
exception is found in the elasmobranches, which produce 10: hydroxycorticosterone as
their major glucocorticoid (Idler and Truscott 1967).

The mineralocorticoids are characterized by a hydroxyl at the carbon 11 position,
and the carbon 18 oxidized to an aldehyde (Norman and Litwack 1997). The principal
mineralocorticoid is aldosterone in land vertebrates which is important in regulating ion

exchange and water metabolism. In ﬁsh, there is no evidence that aldosterone exists. It

was thought that cortisol had a dual role as stress steroid and mineralocorticoid in ﬁsh.
However, a recent study has found that ll-deoxycorticosterone was an agonist for the
mineralocorticoid receptor in rainbow trout (Sturm et al. 2005).

Glucocorticoids are considered stress steroid hormones with many functions.
Sapolsky et al. (2000) classiﬁed the roles of glucocorticoids in the stress response as
permissive, suppressive, stimulatory, and preparative. The suppressive functions of
glucocorticoids include regulation of reproduction, inﬂammation, and immune system,
while permissive actions are important in adrenal gland insufﬁciencies or malﬁmctions
(Sapolsky et al. 2000). Stimulatory functions of glucocorticoids include regulating
metabolic processes like prolonged elevation of blood glucose by gluconeogenesis and

inhibition of glucose utilization (Sapolsky et al. 2000).

The sea lamprey as a model system

The sea lamprey (Petromyzon marinus) offers a unique animal model for the
studies of stress steroid hormone characterization due to its ancestral lineage. The
lampreys are Agnathans, jawless ﬁshes, which are one of the oldest extant vertebrates
available for use in the characterization of stress steroid hormones. Characterizing the
glucocorticoid in one of the earliest vertebrates helps us understand the evolution of
stress hormones in the vertebrate lineage. While we know that most vertebrates utilize
cortisol or corticosterone as their glucoccorticoid, it was not known whether these ancient

j awless ﬁsh possess a stress steroid.

Another beneﬁt in using lamprey as a model is that the stress steroid hormone
should be available at different life history stages of the lamprey. During the life cycle of
the lamprey, they experience a freshwater and saltwater phase. These different phases
include changing environments that may expose the lamprey to different stressors. The
larvae go through a transformation called metamorphosis to prepare for the ocean or lake
phase of their life cycle. After the parasitic phase, the sea lampreys reenter freshwater to
spawn in tributaries where the eggs hatch in nests. The larvae leave the nests and then
drift into soft substrate areas and reside as larvae for several years. The process of
metamorphosis shares similar characteristics of smolting in salrnonids. It is well known
that sahnonid smoltiﬁcation is regulated by glucocorticoid cortisol. Therefore, lamprey
may have a glucocorticoid hormone that acts similar to cortisol. The parasitic and
spawning phases of the adult lampreys should also have a glucocorticoid stress response
to help maintain allostasis.

The sea lamprey also offers a good model to understand the glucocorticoid stress
response for ﬁsheries management purposes. Sea lampreys are an undesirable pest in the
Laurentian Great Lakes. Since the 19505, substantial amounts of funding and effort to
control the sea lamprey population have occurred. However, lamprey populations around
the world are in decline due to habitat destruction. In the Paciﬁc Northwest, the Paciﬁc
lampreys (Entosphenus tridentatus) are important subsistence foods for indigenous
peoples. Identifying the glucocorticoid and being able to measure the stress steroid may
assist in evaluating environmental stressors and various management activities for better

conservation or control.

Introduction to dissertation

There is evidence that lampreys have the primary, secondary, and tertiary stress
responses. For example, it has been shown that lamprey exhibit increases in
catecholamines after an acute stressor, which is part of the primary stress response
(Mazeaud and Mazeaud 1981). Plasma levels of epinephrine and norepinephrine were
shown to increase in sea lamprey after various stressors (Mazeaud and Mazeaud 1981).
The Hypothalamus-Pituitary-Adrenal (interrenal) Axis may exist in lamprey. While there
is no evidence for CRH from the hypothalamus, there is evidence for ACTH in the
lamprey pituitary. ACTH from the lamprey pituitary has been cloned and sequenced
(Heinig et al. 1995; Takahashi et al. 1995A; Takahashi et a1. 1995B). The lamprey
ACTH is 60 amino acids long, roughly 20 amino acids longer than other vertebrate
ACTHs (Takahashi et al. 2006). However, lamprey ACTH levels have not been shown
to increase after acute stress. In addition, there is no direct evidence for a stress steroid
hormone or glucocorticoid in the j awless vertebrate, the sea lamprey.

There is evidence that lampreys exhibit a secondary stress response. Different
types of stressors such as dewatering and handling can induce hyperglycemia in lampreys
(Larsen 1976; Morris and Islam 1969; Leibson and Plisetskaya 1969). Close et al. (2003)
provided evidence that handling and tagging can change indicators of secondary and
tertiary stress responses such as plasma glucose, ventilation rate, and swimming

performance in lamprey.

The goal of this research was to identify and characterize the stress steroid
hormone in the ancient vertebrate, the sea lamprey. To accomplish this goal, the ﬁrst
prediction was that lampreys have a stress steroid circulating in the plasma (chapter 1).
The second prediction was that any identiﬁed steroids have a receptor (chapter 2). The
third prediction was that identiﬁed steroids that have a receptor will have classic

glucocorticoid stress response and show biological effects (chapter 3).

10

REFERENCES

Bern, HA. 1967. Hormones and endocrine glands of ﬁshes. Science 158:455-462.

Brett, J .R. 1958. Implications and assessments of environmental stress. In Investigations
of Fish-Power Problems. Pages 69-83 in RA. Larkin, editor. H.R. MacMillan
Lectures in Fisheries, University of British Columbia.

Cannon, W.B., and Pereira J .R. 1924. Increase of adrenal secretion in fever. Proceedings
of the National Academy of Sciences 10:247-248.

Cannon, W.B. 1929. Organization for physiological homeostasis. Physiological Reviews
9(3):399-431.

Close, D.A., Fitzpatrick, M.S., Lorion, C.M., Li, H.W., and Schreck, CB. 2003. Effects
of intraperitoneally implanted radio transmitters on the swimming performance and
physiology of Paciﬁc Lamprey. North American Journal of Fisheries Management
23:1184-1192.

Donaldson, EM. 1981. The pituitary-interrenal axis as an indicator of stress in ﬁsh.
Pages 11-47 in AD. Pickering, editor. Stress and ﬁsh. Academic Press, London.

Heinig, J .A., Keeley, F.W., Robson, P., Sower, S.A., and Youson, J .H. 1995. The
appearance of pr00piomelanocortin early in vertebrate evolution: cloning and
sequencing of POMC from a lamprey pituitary Cdna library. General and
Comparative Endocrinology 99: 137-144.

Idler, DR. and Truscott, B. 1967. 1a-Hydroxycorticosterone: synthesis in vitro and
properties of an interrenal steroid in the blood of cartilaginous ﬁsh (Genus Raja).
Steroids 9:457-477.

Idler, DR. and Truscott, B. 1972. Corticosteroids in ﬁsh. Pages 126-252 in DR. Idler,
editor. Steroids in nonmammalian vertebrates. Academic Press, New York.

Jones, I.C., Bellamy, D., Chan, D.K.O., Follett, B.K., Henderson, I.W., Phillips, J .G., and
Snart, RS. 1972. Biological actions of steroid hormones in nonmammalian
vertebrates. Pages 414-480 in DR. Idler, editor. Steroids in nomnammalian
vertebrates. Academic Press, New York.

Landys, M.M., Ramenofsky, M., and Wingﬁeld, J .C. 2006. Actions of glucocorticoids at
a seasonal baseline as compared to stress-related levels in the regulation of periodic
life processes. General and Comparative Endocrinology 148:132-149.

11

Larsen, L0. 1976. Blood glucose levels in intact and hypophysectomized lampreys
(Lampetraﬂuviatilis L.) treated with insulin, “stress,” or glucose, before and during
the period of sexual maturation. General Comparative Endocrinology 29:1-13.

Leibson, LG, and Plisetskaya, EM. 1969. Hormonal control of blood sugar level in
cyclostomes. General Comparative Endocrinology 2:528-534.

Mazeaud, M.M., Mazeaud, F., and Donaldson, EM. 1977. Primary and secondary effects
of stress in ﬁsh: some new data with a general review. Transactions of the American
Fisheries Society 106:201-212.

Mazeaud, M.M., and Mazeaud, F. 1981. Adrenergic responses to stress in ﬁsh. Pages 49-
75 in AD. Pickering, editor. Stress and ﬁsh. Academic Press, London.

McEwen, BS. 2000. Allostasis and allostatic load: Implications for
neuropsychopharrnacology. Neuropsychopharmacology 22: 108-124.

McEwen, BS, and Stellar, E. 1993. Stress and the individual. Archives of Internal
Medicine 153:2093-2101.

McEwen, BS, and Wingﬁeld, J.C. 2003. The concept of allostasis in biology and
biomedicine. Hormones and Behavior 43:2-15.

Mommsen, T.P., Vijayan, M.M., and Moon, T.W. 1999. Cortisol in teleosts: dynamics,
mechanisms of action, and metabolic regulation. Reviews in Fish Biology and
Fisheries 92211-268.

Morris, R., and Islam, D.S. 1969. The effect of hormones and hormone inhibitors on
blood sugar regulation and the follicles of Langerhans in ammocoete larvae. General
Comparative Endocrinology 12:8 1-90.

Munck, A., Guyre, P.M., and Holbrook, NJ. 1984. Physiological ﬁmctions of
glucocorticoids in stress and their relations to pharmacological actions. Endocrine
Reviews 5:25-44.

Norman, A.W. and Litwack, G. 1997. Hormones. 2nd edition. Academic Press, San
Diego.

. Pickering, AD. 1981. Introduction: the concept of biological stress. Pages 1-9 in AD.
Pickering, editor. Stress and ﬁsh. Academic Press, London.

Robin, ED. 1979. Claude Bernard and the internal environment. A memorial
symposium. Marcel Dekker, New York.

12

Sapolsky, R.M., Romero, L.M., and Munck, A.U. 2000. How do glucocorticoids
inﬂuence stress responses? Integrating permissive, suppressive, stimulatory, and
preparative actions. Endocrine Reviews 21 :55-89.

Sandor, T. 1972. Corticosteroids in amphibia, reptilia, and aves. Pages 253-327 in DR.
Idler, editor. Steroids in nonmammalian vertebrates. Academic Press, New York.

Sayers, G. 1950. The adrenal cortex and homeostasis. Physiological Reviews 30:241-320.

Schreck, CB. 1981. Stress and compensation in teleostean ﬁshes: response to social and
physical factors. Pages 295-321 in AD. Pickering, editor. Stress and ﬁsh. Academic
Press, London.

Schreck, CB, and Li, H.W. 1991. Performance capacity of ﬁsh: stress and water quality.
In D.E. Brune and J .R. Tomasso, editors. Aquaculture and water quality. World
Aquaculture Society, Baton Rouge, LA. Advances in World Aquaculture 3:21-29.

Sterling, P. and Eyer, J. 1988. Allostasis: a new paradigm to explain arousal pathology.
Pages 629-649 in S. Fisher and J. Reason, editors. Handbook of life stress, cognition
and health. John Wiley & Sons, New York.

Selye, H. 1936. A syndrome produced by diverse nocuous agents. Nature 138:32.

Selye, H. 1946. The general adaptation syndrome and the diseases of adaptation. The
Journal of Clinical Endocrinology 62117-230.

Selye, H. 1950. Stress and the general adaptation syndrome. British Medical Journal
121383-1392.

Selye, H. 1956. The stress of life. McGraw-Hill, New York.
Selye, H. 1973. The evolution of the stress concept. American Scientist 61 :692-699.

Sturm, A., Bury, N., Dengreville, L., F agart, J ., Flouriot, G., Rafestin-Oblin, ME, and
Prunet, P. 2005. 11-Deoxycorticosterone is a potent agonist of the rainbow trout
(Oncorhynchus mykiss) mineralocorticoid receptor. Endocrinology 146:47-55.

Takahashi, A., Amemiya, Y., Sarashi, M., Sower, S.A., and Kawauchi, H. 1995.
Melanotropin and corticotropin are encoded on two distinct genes in the lamprey, the
earliest evolved extant vertebrate. Biochemical and Biophysical Research
Communications 2 13:490-498.

Takahashi, A., Amemiya, Y., Nozaki, M., Sower, S.A., Joss, J ., Gorbman, A., and

Kawauchi, H. (1995). Isolation and characterization of melanotropins from lamprey
pituitary glands. International Journal of Peptide & Protein Research 46: 197-204.

13

Takahashi, A., Yasuda, A., Sower, S.A., Kawauchi, H. 2006. Posttranslational processing
of pr00piomelanocortin family molecules in sea lamprey based on mass spectrometric
and chemical analysis. General and Comparative Endocrinology 148:79-84.

Wedemeyer, G.A., and McLeay, DJ. 1981. Methods for determining the tolerance of
ﬁshes to environmental stressors. Pages 247-275 in AD. Pickering, editor. Stress and
Fish. Academic Press, London.

Wedemeyer, G.A., Barton, B.A., and McLeay, DJ. 1990. Stress and acclimation. Pages
451-489 in CB. Schreck and PB. Moyle, editors. Methods for ﬁsh biology.
American Fisheries Society, Bethesda, Maryland.

Wendelaar Bonga, SE. 1997. The stress response in ﬁsh. Physiological Reviews 77:591-
625.

14

CHAPTER 1

IDENTIFICATION OF PUTATIVE GLUCOCORTICOIDS IN THE SEA

LAMPREY Petromyzon marinas

15

 

ABSTRACT

This study was undertaken to identify putative glucocorticoids in the blood of the
sea lamprey Petromyzon marinas. Most glucocorticoid chemical structures are known
throughout the vertebrate lineages. However, previous studies have failed to provide
deﬁnitive evidence that the lamprey, one of the most ancient vertebrates, possess a stress
steroid hormone. We hypothesized that lampreys do have a stress steroid hormone in
their blood. High Performance Liquid Chromatography (HPLC) was used to fractionate
extracted lamprey plasma for screening. Radioimmunoassays and protein binding assays
were used to screen fractionated lamprey plasma for cortisol-like and corticosterone-like
steroids. Radioimmunoassays were also used to monitor steroids in conjunction with
LH-20 partition chromatography followed by HPLC to fractionate 800 ml of extracted
lamprey plasma. Corresponding immunoreactive peaks were collected and subjected to
mass spectrometry. The blood of the lamprey contained possible stress steroid hormones.
Deﬁnitive identiﬁcation was made by mass spectrometry analysis of 1 1-deoxycortisol
and 1 1-deoxycorticosterone circulating in the blood of lamprey. This study provided
evidence that one of these steroids is a putative glucocorticoid in the ancient vertebrate,

the sea lamprey.

16

INTRODUCTION

Corticosteroids are steroid hormones produced by the adrenal cortex in mammals
and the interrenal tissue located in the anterior portion of the kidney in teleosts (Bern
1967; Frazer 1992). Corticosteroids are divided into two groups, the glucocorticoids
(GC) and mineralocorticoids (MC) based on ﬁmction. GCs are considered stress steroid
hormones with many functions. Sapolsky et al. (2000) classiﬁed the roles of GCs in the
stress response as permissive, suppressive, stimulatory, and preparative. The suppressive
functions of glucocorticoids include regulation of reproduction, inﬂammation, and
immune system, while permissive actions are important in adrenal gland insufﬁciencies
or malﬁmctions (Sapolsky et al. 2000). Stimulatory functions of glucocorticoids include
the regulation of metabolic processes like prolonged elevation of blood glucose by
gluconeogenesis and inhibition of glucose utilization (Sapolsky et al. 2000).

The principal stress steroids (GCs) in vertebrates are cortisol or corticosterone
(Bern 1967; Idler and Truscott 1972; Sandor 1972; Jones et al. 1972). Mammals secrete
cortisol; while rats and mice secrete corticosterone. Reptiles and birds also produce and
circulate corticosterone as their primary glucocorticoid (Nelson 2000). In bony ﬁsh, the
active GC is cortisol (Idler and Truscott 1972) while in elasmobranchs like sharks and
rays the active GC is 101- hydroxycorticosterone (Idler and Truscott 1967). In teleosts,
cortisol may act as both a GC and MC which provides homeostasis in salt and water

balance. The principal MC in land vertebrates is aldosterone, which ﬁmctions to regulate

17

sodium and potassium transport and controls water reabsorption in the kidney (Norman
and Litwack 1997). There is no evidence that ﬁsh have aldosterone.

The current thinking is that the steroid/receptor complex evolved in the
vertebrate lineage (Escriva et al. 1997; Baker 1997). The question of whether stress
steroids exist and ﬁmction in ancient j awless ﬁshes had not been answered. Identifying
the possible stress steroids in lamprey would be an important step toward understanding
vertebrate steroid/receptor evolution.

Previous studies have provided some evidence that a stress steroid may exist in
the jawless vertebrates. The ﬁrst line of evidence was from in vitro experiments using
presumptive adrenal tissue incubations (PAT) with tritiated steroid precursors to examine
possible corticosteroids in sea lamprey, Petromyzon marinus. Weisbart and Youson
(1975) incubated PAT from larval and parasitic adult sea lamprey with [4-14C]
progesterone and identiﬁed the products using thin layer chromatography and
recrystallization. The identiﬁed steroids were ll-deoxycortisol, 17oz-
hydroxyprogesterone, and androstenedione. From incubations with testes, the authors
also identiﬁed 11-deoxycorticosterone. The PAT incubations failed to form cortisol,
cortisone, corticosterone, or ll-deoxycorticosterone. Weisbart et al. (1978) conducted
another PAT incubation experiment with [1, 2- 3 H] cholesterol, which failed to produce
any known putative corticosteroids in spawning phase adult sea lamprey. Efforts to ﬁnd
possible corticosteroids in lamprey also included in vivo studies.

The second line of evidence was from in vivo experiments designed to examine
possible corticosteroids in the blood of lamprey. Using double-isotope derivative

methods, it was determined that either lampreys had no corticosteroids or levels were too

18

low to be biologically active (Weisbart and Idler 1970). Weisbart and Idler (1970)
measured plasma cortisol levels at < 5 ng/ 100 m1 , while cortisone and corticosterone
levels were at 2 ng/100 ml in sea lamprey. Another study using the same methods found
serum levels of cortisol, ll-deoxycortisol, corticosterone, at < 3 ng/ 100 ml and cortisone
< 5 ng/ 100 ml in lamprey (Buus and Larsen 1975). Weisbart and Youson (1977) used
chromatography and recrystallization to identify ll-deoxycorticosterone after [3 H]
progesterone injections into parasitic adult sea lampreys. Weisbart et al. (1980) identiﬁed
cortisol, corticosterone, 1 1-deoxycortisol, l 1-dehydrocorticosterone by double-isotope
derivative assay in sea lamprey after injecting porcine adrenocorticotropin releasing
hormone (ACTH). The study failed to identify cortisone or ll-deoxycorticosterone in
lamprey.

The third line of evidence was from recent receptor research in lamprey.
Thornton (2001) identiﬁed DNA sequences which were homologues to estrogen,
progesterone, and corticosteroid receptor in the lamprey. The analysis predicted that
estrogen receptor evolved ﬁrst, followed by the progesterone receptor, then the
corticosteroid receptor. Recently, Bridgham et al. (2006) synthesized the ligand binding
domain of the ancestral corticoid receptor for use in a transactivation assay. The assay
showed that aldosterone and ll-deoxycorticosterone had the highest activation levels of
the luciferase reporter. In addition, Bridgham et al. (2006) provided phylogenetic
evidence that the ancestral corticoid receptor was a mineralocorticoid receptor which then
gave rise to the glucocorticoid receptor in teleosts and tetrapods. The three lines of
evidence mentioned, give reason to postulate that lampreys possess a stress steroid

hormone.

l9

The aim of the study was to establish chemical identiﬁcation of possible stress
steroids present in the plasma of sea lamprey. Our ﬁrst objective was to use
radioimmunoassays to screen lamprey plasma for cortisol-like and corticosterone-like
steroids. Our second objective was to extract the steroids from a large pool of plasma,
then to isolate them using chromatography methods. Our third objective was to
determine the chemical identiﬁcation of the putative stress steroids by mass spectrometry

analysis.

20

MATERIALS AND METHODS

Maintenance of animals and blood collection

Sea lamprey Petromyzon marinas were collected in landlocked streams by the
US. Fish and Wildlife Service employees, and transported to Michigan State University
(East Lansing, MI) or USGS Hammond Bay Biological Station (Millersburg, MI) for
experiments and collecting plasma where they were held at 10-13 °C. Blood was
obtained by cardiac puncture using vacutainers containing EDTA (Becton Dickinson,
Franklin Lakes, NJ), placed on ice for 15 min, and then centrifuged at 1000 x g at 4 ° C
for 15 min. The plasma was removed and stored at -80 °C. All experiments were
approved by the Michigan State University Institutional Animal Care and Use Committee

(AUF # 05/04—077-00).

Plasma Extraction

Pooled plasma (20 ml) from male and female lampreys was diluted 1:1 with 0.9%
saline, passed through a 0.45 p.M ﬁlter (Millipore, Billerica, MA), and loaded onto an
activated Sep-Pak (Waters, Milford, MA). The Sep-Pak was washed with 5 ml deionized
water and eluted with 5 ml methanol. The methanol elute was evaporated under reduced

pressure using a CentriVap Concentrator (Labconco, Kansas City, MO).

21

High performance liquid chromatography

Samples were dissolved in 1 ml actonitrile/water/triﬂuoroacetic acid (TFA)
(28/72/0.01, v/v/v) and loaded onto a C18 reverse-phase HPLC column (Nova-Pak, 3.9
mm x 300 mm, Waters) ﬁtted with a guard module. Two solvents were used to deliver a
gradient to the column. Solvent A was 0.01% TF A in deionized water and solvent B was
70% acetonitrile and 0.01% TFA in deionized water. The pattern of development was as
follows: 0 —. 10 min, 28% B; 10 -> 60 min, 28 -> 100% B; 60 -’ 80 min, 100% B. The
eluate was monitored for UV absorption with a photodiode array detector (Waters).

Fractions were collected every 1 min between 11 and 70 min into 1.5 ml tubes.

Radioimmunoassay (RIA) and protein binding assay (PBA) procedures

RIAs and PBA were conducted in glass culture tubes (10 mm X 75mm, Fisher
Scientiﬁc, Pittsburgh, PA). Brieﬂy, the assay buffer consisted of 50 mM sodium
phosphate, pH 7.4, 0.2% BSA, 137 mM NaCl, 0.40 mM EDTA, and 0.77 mM sodium
azide. Nine standards were made up in duplicate over the range 1.95-500 pg/ 100 til/tube.
The tubes containing samples also had a volume of 100 pl of RIA buffer. Binding
reagent was made by adding radiolabel (American Radiolabled Chemicals, St. Louis,
MO) and antiserum (Chemicon, Temecula, CA., 1:100) or rabbit sera (Sigma-Aldrich, St.
Louis, MO, 1:40) for PBA to 20 ml of assay buffer in amounts such that, when 100 ul
was dispensed to all tubes, each tube contained 5000 dpm and, in the absence of any

standard, 50% of the radiolabel was bound to the antiserum or cortisol binding protein.

22

Blank tubes, to which no antibody was added, and tubes necessary to determine the total
and maximum dpm counts were also included in the assay. All tubes were incubated
overnight at 4 °C. After overnight incubation, 500 pl of ice-cold charcoal solution at 0
°C (50 mM sodium phosphate, Pb 7.4, 0.1% gelatin, 1.0% dextran-coated charcoal) was
added to each tube. The tubes were kept on ice for 20 min, and then centrifuged in an
Allegra 6R (Beckrnan Coulter, Fullerton, CA) at 1000 x g for 12 min. The supernatants
were poured into 8 ml scintillation vials, mixed with 6 ml scintillation ﬂuid and DPM

were counted with an LS-6500 (Beckrnan Coulter, Fullerton, CA) scintillation counter.

LH-20 column chromatography

Sample was dissolved in 2 ml 98% dichloromethane and 2% methanol and loaded
onto a glass column (450 mm x 15 mm) packed with 20.0 g of Sephadex LH-20
(Amersham, Piscataway, NJ). Two solvents were used to deliver sample through the
column. A solution containing 98% dichloromethane and 2% methanol was pumped
through the column at 4 ml/min. Fractions were collected every 1 min between 1 and 60
min into 16 x 100 mm glass culture tubes. Elute was dried down and resuspended with

0.5 ml methanol per tube.

Mass spectrometry analysis of plasma

LH-20 fractions corresponding to immunoreactivity of 11-deoxycortisol and 11-

deoxycorticosterone were collected and each group combined and dried down under

23

reduced pressure. Samples were then fractionated by HPLC and assayed to identify
immunoreactive peaks in fractions. Immunoreactive fractions were combined, dried
down under reduced pressure and then subjected to Atmospheric Pressure Chemical
Ionization Mass Spectrometry (APCI-MS) analysis. Mass spectra were obtained using a
LCQ-Deca ion trap (Thenno Scientiﬁc, Waltham, MA). The vaporizer temperature was
300 °C and the capillary temperature was 250 °C. Samples were compared against
authentic ll-Deoxycortisol and ll-Deoxycorticosterone standards (Si grna-Aldrich).
Mass spectrometry analysis was performed at the Mass Spectrometry Facility, Research

Technology Support Facility at Michigan State University.

24

RESULTS

To identify possible stress steroids in sea lamprey, plasma was screened for
immunoreactive cortisol and corticosterone. In addition, the plasma was also screened
for binding activity with cortisol binding protein. Plasma immunoreactive cortisol levels
peaked in HPLC fraction 44 at 1561 pg ml'l (Fig. 1A). The peak in fraction 44
corresponds to the retention time of 1 1-deoxycortisol standard. A small immunoreactive
peak occurred at ﬁactions 32/33, where the cortisol standard eluted from the column.
However, the peak was only slightly higher than background levels in other fractions.
Background immunoreactive cortisol levels appeared to gradually rise throughout the
HPLC run.

The cortisol binding protein assay showed results similar to the cortisol
radioimmunoassay (Fig. 1B). The cortisol level markedly increased to 2231 pg ml'1 in
HPLC ﬁaction 44. The ll-deoxycortisol standard eluted from the HPLC column at the
same fraction. Cortisol was not detected in HPLC fractions 32/33. Cortisol binding
activity, unlike immunoreactivity to cortisol antibody, showed much lower background
levels in other fractions.

Irnmunoreactive corticosterone peaked in fraction 50 at 608 pg ml'l (Fig 2).
Plasma levels of corticosterone also peaked in fractions 31, 56/57, and 63. However, no
corticosterone was detected in ﬁ'actions 41/42 where corticosterone standard eluted from
the HPLC column. The major peak at fraction 50 corresponded with 11-

deoxycorticosterone standard.

25

To collect a sufﬁcient amount of steroids needed for identiﬁcation, 800 ml plasma
was extracted with Sep-Pak and the sample was subjected to gel ﬁltration on a Sephadex
LH-20 column to separate the immunoreactive steroids. Eluted LH-20 fractions were
screened with ll-deoxycortisol and 11-deoxycorticosterone RIAs (Fig. 3).
Irnmunoreactive ll-deoxycortisol levels were elevated in fractions 19-28 with a peak of
25 ng ml". Irnmunoreactive ll-deoxycorticosterone levels were elevated in fractions 10-
12 with a peak of 23 ng ml". Each set of fractions was combined, dried down, and run
on HPLC separately. HPLC fractions were screened using 11-deoxycortisol and 11-
deoxycorticosterone RIAs. Irnmunoreactive HPLC fractions corresponding with 11-
deoxycortisol (44) and 1 1-deoxycorticosterone (50) were collected, dried down, and
subjected to mass spectrometry analysis.

HPLC fractions corresponding to immunoreactive ll-deoxycortisol and 11-
deoxycorticosterone were analyzed by APCI-MS in positive mode to observe parent ions.
To conﬁrm the identity of the compounds, the parent ions were subj ected to tandem mass
spectrometry (MS/MS) and compared to authentic ll-deoxycortisol and 11-
deoxycorticosterone ﬁ'agmentation patterns of daughter ions. The MS/MS spectrum of
authentic ll-deoxycortisol [M+H]+ ion at m/z 347, obtained by direct infusion of a 10 pg
ml'1 ll-deoxycortisol standard solution, are shown in ﬁgure (4A). The standard
fragmented at m/z 269, 293, 311, and 329, which matched the fragmented daughter ions
of the plasma sample (Fig. 4B).

The MS/MS spectrum of authentic ll-deoxycorticosterone [M+H]+ ion at m/z
331, fragmented at m/z 295 and 313 (Fig. 5A). The two most abundant ions obtained by

fragmenting the plasma sample [M+H]+ ion, are also at m/z 295 and 313 (Fig. 5B).

26

2000-

 

A Cortisol y-Deoxycortlsol

’5 1500 .
0

E g 1000 -
0 L-

: g ‘

500'

0

 

V U U V U V I I I

1 0 20 30 4O 50 60 70

3000 ' Cortisol 11-Deoxycortisol

l l

Cortisol-like binding
(pg/fraction)

 

 

 

1O 20 30 40 50 60 70
Retention Time (min)

Figure 1-1. Screening of HPLC fractions of lamprey plasma for cortisol-like steroids
using RIA and BPA. (A) Amount of immunoreactive cortisol found in 0.5 ml I-IPLC
fractions ﬁ'om RIA of 20 pl of each fraction and back calculated fraction volume. (B)
Amount of cortisol binding activity found in 0.5 ml HPLC ﬁ'actions from PBA of 20 p1
of each fraction and back calculated to fraction volume. Arrows, show where cortisol and
ll-deoxycortisol standard elute. ir = immunoreactivity.

27

1000'
800 4 Corticosterone 1 1 -Deoxycorticosterone
600'

 

 

 

 

 

ir Corticosterone (pg/fraction)

10 20 3O 4O 50 60 70
Retention Time (min)

Figure 1-2. Screening of HPLC fractions of lamprey plasma for corticosterone-like
steroids using RIA. Amount of immunoreactive corticosterone found in 0.5 ml HPLC
fractions from RIA of 20 pl of each fraction and back calculated to fraction volume.
Arrows, show where corticosterone and ll-deoxycorticosterone elute. Ir =
immunoreactivity.

28

30 - —O— 1 1 -Deoxycorticosterone
. + 1 1 -Deoxycortisol

-¥ N N
0| O 01
o

teroids (nglfraction)
8

0|

 

"'8

O

 

 

v I V I v '

0 10 20 3O 40
Retention time (min)

Figure 1-3. Screening of LH-20 fractions of lamprey plasma for immunoreactive 11-
deoxycortisol and ll-deoxycorticosterone by RIA. Amount of immunoreactive 11-
deoxycortisol and ll-deoxycorticosterone found in LH-20 fractions from a Sep-Pak
extract of 800 ml of sea lamprey plasma based on RIA of 20 p1 of each fraction and back
calculated to fraction volume. Ir = immunoreactivity.

29

1 00 . 311,0 329.0
A Authentic 1 1 -Deoxycortisol

 

 

 

 

 

 

 

 

 

50 r
8 ‘ 293.0 347.1
5 . 26 .o
'D 1
g 4
.a .
< o v '. r '1 . . v . . In.“ I - Jr 1 I - '
g 100 150 200 250 300 350
3.3 100 . 329.0
7, i B Plasma sample
a l
i 311.1
3 3417.0
503
1 269.1 293-1
0 ‘ r v t . ' n 'i .1; :J...u lili.J: Ii.v

 

 

 

 

 

 

 

100 150 200 250 300 350
m/z

Figure 1-4. Identiﬁcation of 1 l-deoxycortisol by APCI MS/MS analysis. Fragmentation
patterns generated from both the synthetic ll-deoxycortisol (A) and a compound in
HPLC fraction 44 (B), positively identifying the unknown compound in fraction 44 as

1 l-deoxycortisol.

30

 

 

 

 

 

 

 

 

 

 

 

 

 

100 - 313.1
‘ A Authentic 11-Deoxycorticosterone
- 912°” 295.1
1 C=O
1 331.1
50 1
1 O
4’ 1
U .
C .
a 1
.2 o J v j v ' I 4 NJ. .l.lv 1 - I
3 100 150 200 250 300 350
j 100 . 313.1
E - B Plasma sample
16 ‘ 331.1
7’ l
I! 1
so 1
l
4 295.2
0 I v v 4’ I’L v 1 r Iv —'-I-q
100 150 200 250 300 350
m/z

Figure 1-5. Identiﬁcation of 1 l-deoxycorticosterone by APCI MS/MS analysis.
Fragmentation patterns generated from both the synthetic ll-deoxycorticosterone (A) and
a compound in HPLC fraction 50 (B), positively identifying the unknown compound in
fraction 50 as 11-deoxycorticosterone.

31

DISCUSSION

Biological and chemical analysis provided strong evidence for the identiﬁcation
of l l-deoxycortisol and ll-deoxycorticosterone. There were three lines of evidence to
identify these putative stress steroids in the plasma of the sea lamprey. First, the results
showed co-elution of immunoreactive cortisol, cortisol binding protein activity, and
immunoreactive corticosterone peaks with ll-deoxycortisol and 11-deoxycorticosterone
standards in I-IPLC fractions. Second, the results showed the use of immunoreactive ll-
deoxycortisol and ll-deoxycorticosterone to monitor LH-20 and HPLC fractionation
with imunoreactive peaks corresponding to elution times of 1 l-deoxycortisol and 11-
deoxycorticoserone standards. Third, the study conﬁrmed the identity of the parent ions
of 11-deoxycortisol [M+H]+ ion at m/z 347 and ll-deoxycorticosterone [M+H]+ ion at
m/z 331 from plasma of sea lamprey. To conﬁrm the identity, the parent ions were
subjected to MS/MS analysis to observe the fragmentation patterns. The fragmentation
patterns of the daughter ions of authentic ll-deoxycortisol and ll-deoxycorticosterone
standards matched the fragmentation patterns from the plasma steroid samples.

In earlier studies, steroid identiﬁcation was problematic due to limitations of the
extant techniques and methods. Sandor and Idler (1972) developed the following criteria
for identiﬁcation of steroids; positive, presumptive, tentative, and suggestive based on the
level of rigor in methodology. Idler and Truscott (197 2) used the criteria to examine
earlier reported corticosteroid identiﬁcations. They reported that Weisbart and Idler
(1970) had used; thin layer chromatography (TLC), derivative methods (Der),

recrystallization (Cry), and double-isotope derivative assay (DIDA) to identify cortisol

32

with plasma levels at 50 pg/ml in sea lamprey. In contrast, they reduced the number
techniques to TLC, Der, and DIDA to identify plasma cortisone and corticosterone 20
pg/ml in the sea lamprey (Weisbart and Idler 1970). Thus based on Idler and Truscott’s
identiﬁcation criteria, Weisbart and Idler’s (1970) identiﬁcation of cortisol was a
presumptive identiﬁcation, while cortisone and corticosterone were suggestive
identiﬁcations.

In later studies through the 19705, in vivo and in vitro studies continued using the
same techniques mentioned above to conﬁrm whether lampreys possessed corticosteroids
(Buus and Larsen 1975; Weisbart and Youson 1975; Weisbart and Youson 1977;
Weisbart et al. 1978; Weisbart et al. 1980). These studies could not take the
identiﬁcation criteria beyond presumptive. In the most recent study, Weisbart et a1.
(1980) identiﬁed cortisol (130 pg/ml), 11-deoxycortisol (570 pg/ml), and corticosterone
(210 pg/ml) in the serum of adrenocorticotropin releasing hormone (ACTH) injected sea
lampreys. Although the study reported identifying ll-deoxycortisol, their study also
reported cortisol and corticosterone which is in contrast with our study.

In our study, unlike earlier studies, cortisol and corticosterone were absent in the
circulation of sea lamprey. After fractionating the lamprey plasma by HPLC, only
background levels of immunoreactive cortisol in fractions 32/33 were observed. In
addition, the cortisol binding protein activity showed no binding activity in the ﬁactions
32/33 where cortisol standard eluted. Irnmunoreactive corticosterone was not observed in
fractions where corticosterone standard eluted in HPLC fractions. Even though previous
studies (Weisbart and Idler 1970; Buus and Larsen 1975; and Weisbart et al. 1980)

presumptively identiﬁed low levels of cortisol and corticosterone in the circulation of

33

lamprey, their methods may have been limited by their levels of detection and possibly
background noise.

In the current study, in vitro PAT incubation experiments were conducted to
reproduce the earlier ﬁndings by Weisbart and Youson (1975). Incubation experiments
with [3 H] pregnenolone and [3 H] progesterone with parasitic and spawning phase sea
lamprey presumptive adrenal tissue (data not shown). A major radioactive peak was
observed to co-elute with ll-deoxycortisol standard in the HPLC fractions. The isolated
radioactive peak was subjected to TLC. The radioactive peak co-migrated with 11-
deoxycortisol standard on TLC. However, changing the solvent system caused the
unknown radioactive peak and 11-deoxycortisol to clearly separate. Therefore, the
conclusion was that presumptive adrenal tissue was not the source of 11-deoxycortisol.
These results are in contrast with the previous report that ll-deoyxcortisol was produced
in the sea lamprey PAT (Weisbart and Youson 1975).

In our study, results from RIA, HPLC, LH-20 chromatography, and mass
spectrometry analysis demonstrate strong evidence that ll-deoxycortisol and 11-
deoxycorticosterone are putative glucocorticoids in sea lamprey. In addition, the results
show that cortisol and corticosterone are absent in the plasma of the ancient vertebrate.
Weisbart and Youson (197 5) suggested that the 1 16-hydroxylase may not be present in
lamprey. Our study provides evidence that llﬁ-hydroxylase did not evolve in the steroid
biosynthetic pathway in early vertebrates, but evolved later in the elasmobranch lineage,
since they produce la—hydroxycorticosterone (Idler and Truscott 1967).

In conclusion, this study provided deﬁnitive identiﬁcation of two putative

glucocorticoid hormones in sea lamprey. Although the results provide identiﬁcation of

34

ll-deoxycortisol and ll-deoxycorticosterone in the blood, there is no evidence that these
steroids have receptors or biological functions. The positive identiﬁcation of these two
steroids will allow further testing to determine whether or not each has associated
receptors. In order to classify these steroids as hormones, they must circulate in the
blood, have cognate receptors, and have biological effects. Therefore future research
efforts will focus on testing the ability of 1 1-dexoycortisol and ll-deoxycorticosterone

binding afﬁnity to receptor in lamprey.

35

ACKNOWLEDGEMENTS

A special thanks to the staff of the US. Geological Survey, Hammond Bay
Biological Station for supplying laboratory space and sea lampreys. A special thanks to
Lijun Chen for her assistance with mass spectrometry and the MSU mass spectrometry
facility. This research was supported and funded by the Bonneville Power
Administration. A warm thanks for the support from the Confederated Tribes of the

Umatilla Indian Reservation.

36

REFERENCES

Baker, ME. 1997. Steroid receptor phylogeny and vertebrate origins. Molecular and
Cellular Endocrinology 135: 101 -107.

Bern, HA. 1967. Hormones and endocrine glands of ﬁshes. Science 158:455-462.

Bridgham, J .T., Carroll, S.M., and Thornton, J.W. 2006. Evolution of hormone-receptor
complexity by molecular exploitation. Science 312:97-101.

Buus, O., and Larsen, L0. 1975. Absence of known corticosteroids in blood of river
lampreys (Lampetraﬂuviatilis) after treatment with mammalian corticotropin.
General and Comparative Endocrinology 26:96-99.

Escriva, H., Saﬁ, R., Hanni, C., Langlois, M-C., Saurnitou-Laprade, P., Stehelin, D.,
Capron, A., Pierce, R., and Laudet, V. 1997. Ligand binding was acquired during
evolution of nuclear receptors. Proceedings of the National Academy of Sciences
94:6803-6808.

Fraser, R. 1992. Biosynthesis of adrenal steroids. Pages 117-130 in V.H.T. James, editor.
The adrenal gland, second edition. Raven Press, New York.

Idler, DR. and Truscott, B. 1967. la-Hydroxycorticosterone: synthesis in vitro and
properties of an interrenal steroid in the blood of cartilaginous ﬁsh (Genus Raja).
Steroids 92457-477.

Idler, DR. and Truscott, B. 1972. Corticosteroids in ﬁsh. Pages 126-252 in DR. Idler,
editor. Steroids in nonmammalian vertebrates. Academic Press, New York.

Jones, CL, Bellamy, D., Chan, D.K.O., Follett, B.K., Henderson, I.W., Philips, J .G., and
Snart, RS. 1972. Biological actions of steroid hormones in nonmammalian
vertebrates. Academic Press, New York.

Nelson, R.J. 2000. An introduction to behavioral endocrinology. Sinauer Associates,
Sunderland, Massachusetts.

Norman, A.W., and Litwack, G. 1997. Hormones. Academic Press, San Diego, CA.

Sandor, T. 1972. Corticosteroids in amphibia, reptilia, and aves. Pages 253-327 in DR.
Idler, editor. Steroids in nonmammalian vertebrates. Academic Press, New York.

Sandor, T. and Idler, DR. 1972. Steroid methodology. Pages 126-252 in DR. Idler,
editor. Steroids in nonmammalian vertebrates. Academic Press, New York.

37

Sapolsky, R.M., Romero, L.M., and Munck, A.U. 2000. How do glucocorticoids
inﬂuence stress responses? Integrating permissive, suppressive, stimulatory, and
preparative actions. Endocrine Reviews 21:55-89.

Thornton, J .W. 2001. Evolution of vertebrate steroid receptors from an ancestral estrogen
receptor by ligand exploitation and serial genome expansions. Proceedings of the
National Academy of Sciences 98:5671-5676.

Weisbart, M., and Idler, DR. 1970. Re-exarnination of the presence of corticosteroids in
two cyclostomes, the Atlantic hagﬁsh (Myxine glutinosa L.) and the sea lamprey
(Petromyzon marinas L.). Journal of Endocrinology. 46:29-43.

Weisbart, M., and Youson, J .H. 1975. Steroid formation in the larval and parasitic adult
sea lamprey, Petromyzon marinas L. General and Comparative Endocrinology
27:5 1 7-526.

Weisbart, M., and Youson, J .H. 1977. In vivo formation of steroids from [1,2,6,7-3H]
progesterone by the sea lamprey, Petromyzon marinas L. J 01mm] of Steroid
Biochemistry 8: 1249-1252.

Weisbart, M., Youson, J .H., and Weibe, J .P. 1978. Biochemical, histochemical, and
ultrastructural analyses of presumed steroid-producing tissues in the sexually mature
sea lamprey, Petromyzon marinus L. General and Comparative Endocrinology 34:26-
37.

Weisbart, M., Dickhoff, W.W., Gorbman, A., and Idler, D. 1980. The presence of

steroids in the sera of the Paciﬁc hagﬁsh, Eptatretus stouti, and the sea lamprey,
Petromyzon marinas. General and Comparative Endocrinology 41 :506-519.

38

CHAPTERZ

CHARACTERIZATION OF A GLUCOCORTICOID RECEPTOR IN THE GILL

OF THE SEA LAMPREY Petromyzon marinas

39

ABSTRACT

This study was conducted to determine if 11-deoxycortisol or 11-
deoxycorticosterone identiﬁed in chapter 1 had a receptor in the gill tissue of the sea
lamprey Petromyzon marinas. Previous research has provided evidence that a
corticosteroid receptor may exist in an ancient vertebrate. This prompted the hypothesis
that one of the two possible corticosteroids; ll-deoxycortisol and ll-deoxycorticosterone
identiﬁed must have a cognate receptor in the gill cytosol of sea lamprey. This study
identiﬁed and characterized high afﬁnity (Kd = 2.66 i 0.47 nM, mean :t SEM, n = 3), low
capacity (Bmax = 58.10 i 3.33 fmollmg protein, n = 3) glucocorticoid receptors in the sea
lamprey gill cytosol. The receptor appears to be highly speciﬁc for 1 1-deoxycortisol.
Competitive binding assays showed that only 11-deoxycortisol displaced [3H] 11-
deoxycortisol compared to nine other steroids. The glucocorticoid receptor had the
highest speciﬁc 11-deoxycortisol binding activity in the intestine followed by gill, testis,
liver, kidney, heart, and muscle. The speciﬁc ll-deoxycortisol-receptor complex binds
to DNA-cellulose. These results are consistent with characteristics of glucocorticoid

receptors in ﬁsh.

40

INTRODUCTION

The nuclear receptor (NR) superfamily arose very early in the evolutionary
history of animals (Laudet 1997; Escriva et al. 1997). Recent phylogenetic studies
suggest that NRs evolved sometime between the Metazoan and Bilateria divergence
(Thornton 2003a; Bertrand et al. 2004). The superfamily receptor groups have the
characteristic of binding to small molecules such as steroids, thyroid hormones, fatty
acids, and retinoic acids (Robinson-Rechavi et al. 2003). The NR superfamily consists of
1i gand-dependant transcription factors that provide the bridge between hydrophobic
signaling hormones and target gene expression (Bertrand et al. 2004).

The steroid receptor subgroup was thought to be restricted to vertebrates (Escriva
et al. 1997; Baker 1997); however, recent research has shown that steroid receptors
similar to vertebrate estrogen receptors exist in mollusks (Thornton et al. 2003b; Keay et
al. 2006). While chordate estrogen receptors have ligands that are signaling hormones,
protostome estrogen receptors were also found, yet it is unclear if they are functional or
not (Keay et al. 2006). It is possible that the mollusks have the estrogen-like receptor but
do not have the enzymes capable of producing estrogens.

The sea lamprey Petromyzon marinas is an agnathan, one of the oldest extant
vertebrates which emerged over 500 million years ago (Kumar and Hedges 1998; Shu et
al. 1999). Thornton (2001), using PCR techniques, ampliﬁed DNA segments from sea
lamprey that were homologues to parts of estrogen, progestin, and corticoid receptor
genes in higher vertebrates. Phylogenetic analysis using the DNA sequences showed

progestin and corticoid receptors evolved from the estrogen receptor. Recently,

41

Bridgham et al. (2006) using maximum likelihood phylogeny and existing ligand binding
domain sequences, inferred the amino acid sequence of the ligand binding domain of the
presumed ancestral corticoid receptor. They also synthesized the sequence of the
corticosteroid ligand binding domain for use in a transactivation assay. The assay
showed that aldosterone and ll-deoxycorticosterone had the highest activation levels of
the luciferase reporter. In addition, Bridgham et al. (2006) provided phylogenetic
evidence that the ancestral corticoid receptor was a mineralocorticoid receptor which then
gave rise to the glucocoricoid receptor in teleosts and tetrapods.

There have been several efforts to characterize steroid receptors from lamprey.
Ho et al. (1987) characterized an estrogen binding moiety in the testes of the sea lamprey.
The putative receptor shared similar binding characteristics to higher vertebrate estrogen
receptor. It was thought that androgen receptor did not exist in lamprey (Thornton 2001).
However, Bryan et al. (2007) provided evidence of an androgen receptor in the sea
lamprey testes. The study suggested that androstenedione was the ligand; however, the
study was unable to convincingly prove that the moiety was not a binding protein
typically found in the gonads. There have been no studies characterizing the
corticosteroid receptor in lamprey.

The aim of this study was to determine if the two deﬁnitively identiﬁed steroids
11- deoxycortisol and ll-deoxycorticosterone in chapter 1 are hormones in lamprey. In
order to classify identiﬁed steroids as hormones, 1) they must be in circulation in the
blood; 2) they must have a receptor, and 3) they must mediate relevant biological effects.
This chapter will focus on whether or not lampreys possess a receptor for either of the

identiﬁed steroids from chapter 1, l 1-deoxycortisol or ll-deoxycorticosterone.

42

MATERIALS AND METHODS

Animals and holding facilities

Sea lamprey Petromyzon marinas were collected in streams by the US. Fish and
Wildlife Service employees, and transported to Michigan State University (East Lansing,
MI) or USGS Hammond Bay Biological Station (Millersburg, M1) for tissue collection.
Lampreys were held at 10-13 °C in ﬂow through tanks. Tissues collected from ﬁsh were
frozen in liquid nitrogen and held at -80 °C until processed for cytosolic fractions. All
experiments were approved by the Michigan State University Institutional Animal Care

and Use Committee (AUF # 05/04-077-00).

Materials

Radiolabled steroids were obtained from American Radiolabled Chemicals (St.

Louis, MO). Synthetic steroids were obtained from Steraloids (Newport, RI) and Sigma

(St. Louis, MO). All other reagents were obtained from Sigma unless otherwise noted.

Preparation of cytosolic fractions of tissues

The decision to use gill cytosol was based on previous research to characterize

glucocorticoid receptors in ﬁsh. It is well known that gill tissues mainly have

glucocorticoid receptors and the receptors are expressed at higher levels than other tissues

43

in teleosts. Preparation of cytosolic fractions of tissues was adapted from Patino and
Thomas (1990). Frozen tissue was ground in liquid nitrogen with a mortar and pestle.
Frozen tissue was mixed 1:5 (weight: volume) in HEPES buffer (25 mM HEPES, 10 mM
NaCl, 1 mM monothioglycerol, pH 7.4) and kept on ice while being homogenized. The
homogenate was centriﬁrged at 1000 x g for 15 min at 4 °C. The supernatant containing
the cytosolic ﬁ'action was collected and the pellet discarded. The supernatant was
centrifuged at 40,000 x g for l h at 4 °C. The supernatant was removed and glycerol

(10% v/v) was added. Cytosolic ﬁactions were immediately used in subsequent assays.

Saturation curve and scatchard analysis

Radiolabled 1 1-deoxycortisol (0.2-20 nM) in ethanol was added to each assay
tube with or without 1 pg cold 1 1-deoxycortisol (to determine non-speciﬁc binding). The
ethanol was evaporated under nitrogen at 40 °C. Then, 200 pl of gill cytosol were added
to the assay tubes and incubated at 0 °C for 2 h. After incubation, 500 pl of ice cold
dextran coated charcoal (DCC) solution were added to assay tubes and incubated on ice
for 5 min. The samples were then centrifuged at 1000 x g for 5 min at 4 °C, and the
supematants were poured into scintillation vials. Six ml of scintillation cocktail were
added to each vial and disintegrations per minute (DPM) were counted by a scintillation
counter. The concentration of binding sites (Bmax) and the dissociation constant (KD)
were determined by hyperbolic regression using Sigrnaplot v9.0 (SYSTAT, San Jose,
CA, USA). Protein concentrations were determined by DC Protein Assay Kit for

microplates (Bio-Rad, Hercules, CA, USA) using bovine serum albumin as a standard.

44

Association and dissociation kinetics

The rate of association was determined by incubating gill cytosol (200 pl) with
3.3 nM of [3 H] ll-deoxycortisol with or without 1 pg of 1 l-deoxycortisol at 0 °C for 0-4
hr. To determine the dissociation rate, gill cytosol (200 pl) was incubated with 3.3 nM of
[3 H] ll-deoxycortisol in the presence or absence of 1pg of l l-deoxycortisol at 0 °C for 2
hr and then the dissociation was initiated by adding 1 pg of 11-deoxycortisol to all assay
tubes for another 0-2 hr at 0 °C. Bound and free steroid were separated by addition of
DCC. The tubes were kept on ice for 5 min, and then centrifuged at 1000 x g for 5 min.
The supematants were poured into 8 ml scintillation vials, mixed with 6 ml scintillation

ﬂuid and DPM were counted using a scintillation counter.
Steroid and tissue binding specificity

To determine steroid speciﬁcity of the binding moiety, cold steroids were used for
their ability to compete with [3 H] ll-deoxycortisol binding. Gill cytosol preparations
(200 p1) were incubated at 0 °C for 2 hr with 3.3 nM [3H] 11-deoxycortisol in the
presence of different amounts of cold steroid (0.1-1000 nM). Speciﬁcity was examined
for ll-deoxycortisol (S), ll-deoxycorticosterone (DOC), cortisol (F), corticosterone (B),
aldosterone (Aldo), dexamethasone (D), androstenedione (Ad), 176-estradiol (E2), 1701-

hydroxyprogesterone (17a-P), and progesterone (P).

45

Relative binding was measured in the gill, intestine, testis, liver, kidney, heart,
and muscle by binding assays. Radiolabled S (5 nM) in ethanol was added to each tube
in the presence or absence of 1pg of cold S and dried down. Each tube received an
aliquot of gill cytosol (200 pl) and was incubated for 2 hr at 0 oC. The reaction was
stopped by the addition of DCC. The tubes were kept on ice for 5 min, and then
centrifuged at 1000 x g for 5 min. The supematants were poured into 8 ml scintillation
vials, mixed with 6 ml scintillation ﬂuid and DPM were counted using a scintillation

counter.

DNA-cellulose chromatography

DNA-cellulose chromatography procedures were modiﬁed from Knoebl et al.
(1996). Hepes buffer with 0.2 mg/ml BSA consisted of three different concentrations of
NaCl, 0.05 M (column buffer), 0.4 M (elution buffer), and 2.0 M (wash buffer). Gill
cytosol (1.0 ml) was incubated for 2 hr at 0 °C with 20 nM [3H] S with or without 2 pg
cold S. Samples were placed on a lab table for 30 min at 25 °C and then cooled back
down with ice for 5 min. The samples were then diluted in 3 ml of column buffer (total
volume 4 ml) and added to a 20-ml column (Bio-Rad, Hercules, CA) containing 5 ml
DNA-cellulose (Amersham, Piscataway, NJ) in column buffer. The sample was allowed
to ﬂow into the DNA-cellulose, and then the ﬂow was stopped to allow absorption for 20
min. The column was then washed with 20 ml of column buffer to remove free

radiolabled steroid. To elute the bound receptor complex from the DNA, 7 ml of 0.4 M

46

NaCl elution buffer followed by 7 ml of wash buffer (2.0 M NaCl) was used. One ml

fractions were collected and DPM were counted using a scintillation counter.

47

RESULTS

Saturation curve and determination of K, and B...“

Saturation binding assays showed speciﬁc and saturable binding moiety for S in
the cytosolic ﬁaction of the gill homogenate (Fig. 1A). Hyperbolic regression analysis
(Fig. 1B) revealed the cytosol binding moiety had a high afﬁnity (Kd = 2.66 3c 0.47 nM,

mean i SEM, n = 3) and low capacity (Bmax = 58.10 i 3.33 ﬁnol/mg protein, 11 = 3) for S.

Association and dissociation kinetics

Kinetic studies of the S binding moiety showed the association rate (Tl/2) was
2.11 i 0.32 min (11 = 3) and the speciﬁc binding remained constant during the experiment
(Fig. 2A). The speciﬁc binding was reversible with a dissociation rate (Tl/2) of 26.44 :t

8.41 min (n = 3) during the 2-hr experiment (Fig. 2B).
Steroid and tissue specificity

S had the highest afﬁnity to the cytosolic binding moiety, among the 9 steroids
tested (Fig. 3). The other steroids failed to displace 50% of 3.3 nM of [3H] S up to 1000

nM concentration. At 1000 nM concentration, DOC almost displaced 50 % of [3H] S

speciﬁc binding. The remaining steroids did not compete for the S binding moiety.

48

Speciﬁc binding of S to the binding moiety was found in all tissue tested (Fig. 4).
The highest levels of speciﬁc binding were found in cytosolic preparations from gill,
intestine, and testis. Relatively low speciﬁc binding was found in the liver, kidney, heart,

and muscle tissues.
DNA-cellulose chromatography

The [3 H] 1 1-deoxycortisol receptor complex showed speciﬁc (BS) DNA binding
in the DNA-cellulose chromatography assays (Fig. 5). The speciﬁc and non-speciﬁc

(BN3) binding were both eluted with 0.40 M NaCl buffer. The 2.0 M NaCl wash buffer

did not elute any additional speciﬁc or non-speciﬁc binding.

49

200001

15000- ' 3N8

 

 

Bound [3H] 11-Deoxycortisol
(rnawn

 

 

1oooo- . °
sooo« .
o ' . . . .
o 5 1o 15 20
[3H] 11-Deoxycortisol (nM)
B so- __________________________________
g 50 . Bmax
3 O
2 40
Q
E 30-
3
E 20
m” 10-
0 . . . . .
o 5 1o 15 20
F(nM)

Figure 2-1. Representative saturation curve (A) and hyperbolic regression plot (B) of 11-
dexoycortisol binding to gill cytosol. The abbreviations are; total binding (BT), speciﬁc
binding (B3), non-speciﬁc binding (BN5), maximum binding capacity of tissue (Bmax),
free (i.e., unbound) [3 H] l 1-deoxycortisol (F), and disintegrations per minute (DPM) are
shown; n = 3.

50

100 ' . o

 

 

 

 

0 60 120 1 80 240

100 4'

80-

60'

Specific [3H] 11-Deoxycortisol binding (%)

40‘

20‘

 

0 . . . . . .
0 20 40 60 80 100 120

Time (min)

 

Figure 2-2. Association and dissociation kinetics of a sea lamprey receptor in gill
cytosol. For association kinetics (A), Tm was 2.11 d: 0.32 min, 11 = 3. For dissociation
kinetics (B), Tm was 26.44 i 8.41 min; 11 = 3.

51

 

 

#r——--"'— --------- v ________ v
0:100 _::-::—:~-—*~—~;_ ._- - iii-'13
: A...” ~~~~~~ """“"' ............... O§§T+ ——————— a
'g 80 . —._ s i ....................... ‘
._ ........ O ........ DOC
.D ——+—- F
.2 so - _.._A._.._ 3
2‘5 ——--— Aldo ..

—'—U—'— D O
8. 40 ' —+— A
U) —<>— E2
°\° 20 d ........ ‘ ........ 17OHP

——-v—-- P

O .

 

 

0.1 1 10 100 1000
Log Competitor Concentration (nM)

Figure 2-3. Speciﬁcity of the lamprey gill cytosolic moiety for various steroids including
ll-deoxycortisol (S), 11-deoxycorticosterone (DOC), cortisol (F), corticosterone (B),
aldosterone (Aldo), Dexamethasone, (D), androstenedione (A), Estradiol (E2), 1701-
hydroxyprogesterone (17 OHP), and progesterone (P); n = 3.

52

701

 

 

 

 

 

 

 

75 60-
'5
'5 5°' 1L
'- —I—
O.
E 40-
: 30 -
o
g 20-
(D
m 10 ' If]
0 " . [“1“] WW—

 

Gill Intestine Testis Liver Kidney Heart Muscle

Tissue

Figure 2-4. Speciﬁc binding of [3 H] ll-deoxycortisol (5 nM) to cytosolic moiety ﬁom
different tissues. Abbreviation; speciﬁc binding (BS). Vertical bars represent means i
SEM; n = 3.

53

   
  

 

 

 

’2‘

n.

9, 2000 0.05 M NaCl , 0.40 M NaCl . 2.0 M NaCl

3 1 ' '

.22 ' '

‘l: 1500 I '

3 . '

g i —e— B... I I

C? . '*‘ 3,, |

g I

.— 500 1 I

m:I:

H ‘ 0"°.0--o.--O"‘O"'¢"“O""O

g o ITTTTTFCJZ-virrr""
o

m 0 5 10 15 20

Fraction

Figure 2- 5. Elution of 1 l-deoxycortisol-receptor complex from DNA-cellulose. Gill
cytosol was preincubated with 20 nM [3H] ll-deoxycortisol before binding to the column
of DNA-cellulose. Abbreviations are; total binding (137), speciﬁc binding (BS),
nonspeciﬁc binding (BN5), and disintegrations per minute (DPM) are shown; 11 = 3.

54

DISCUSSION

This study has provided several lines of evidence to support the hypothesis that
ll-deoxycortisol is a stress steroid and has a glucocorticoid receptor in the gill of an
ancestral vertebrate, the sea lamprey. Our study demonstrated the presence of a
glucocorticoid receptor in the gills of the sea lamprey. The binding moiety was
characterized by high afﬁnity, low capacity glucocorticoid binding sites speciﬁc to 11-
deoxycortisol within gill cytosolic extracts. The dissociation constant (Kd) in our study
falls within the range reported for glucocorticoid receptors using [3 H] cortisol in the gill,
liver, intestine, muscle, leukocytes, erythrocytes and brain of sahnonids (Chakraborti et
al. 1987; Chakraborti and Weisbart 1987; Pottinger 1990; Maule and Schreck 1991;
Knoebl et al. 1996; and Pottinger and Brierley 1997). The concentration of binding sites
(Bmax) in our study was lower than concentrations (224 fmol/mg protein) found in
salmonid gill tissue (Chakraborti et a1. 1987), but was higher than salmonid brain,
intestine, and leukocytes (Chakraborti et al. 1987; Maule and Schreck 1991; Knoebl et al.
1996)

In our study, the glucocorticoid binding moiety shared similar binding
characteristics to glucocorticoid receptors in salmonids (Chakraborti and Weisbart 1987;
Chakraborti et al. 1987). Although, the association and dissociation rates of the lamprey
glucocorticoid receptor were similar to salmonid glucocorticoid receptor, the rates (Tl/2)

were faster. For example, Knoebl et al. (1996) using brain cytosolic extracts estimated

55

the dissociation rate Tm = 64.3 min, compared to T1 /2 = 26.44 min using gill cytosol in
our study.

The sea lamprey corticoid binding moiety is highly speciﬁc to 11-deoxycortisol.
The speciﬁcity of the corticosteroid binding moiety in the gill was different than those
reported for other glucocorticoid receptors found in ﬁsh (Chakraborti et al. 1987;
Chakraborti and Weisbart 1987; Pottinger 1990; Maule and Schreck 1991; Knoebl et al.
1996; and Pottinger and Brierley 1997). In this study, the synthetic steroids
dexarnethasone and triamcinolone acetonide (data not shown), along with natural
corticosteroids, were ineffective at displacing ll-deoxycortisol from the binding moiety.
Chakraborti et al. (1987) found ll-deoxycortisol had the highest afﬁnity of the natural
corticosteroids tested with brook trout (Salvelinusfontinalis) gill receptors followed by
cortisol and corticosterone.

Recently, Bridgeham et al. (2006) using existing corticoid ligand binding domain
sequences, inferred the amino acid sequence of the ligand binding domain of the
presumed ancestral corticoid receptor. They synthesized the ligand binding domain of
the corticoid receptor in lamprey and developed a transactivation assay. Their assay
showed the ligand binding domain to be activated by aldosterone, 11-
deoxycorticosterone, ll-deoxycortisol, corticosterone, and cortisol. The current study
used native receptor complex and found highly speciﬁc binding only for 1 1-deoxycortisol
with limited displacement binding at high concentrations of 1 l-deoxycorticosterone.

The steroid speciﬁcity assay and plasma screening for cortisol and corticosterone
(chapter 1) provides strong evidence that the ancestral vertebrate does not produce

cortisol or corticosterone and thus lacks the 11 ﬂ-hydroxylase enzyme. This could

56

explain why in our study, the native receptor does not recognize cortisol or
corticosterone. The ﬁndings using the native receptor contrast the results of Bridgeham
et al. (2006). However, they used an inferred amino acid sequence and only included the
ligand binding domain of the receptor. This may have changed the protein conformation
creating a nonspeciﬁc sequence allowing other corticosteroids to bind.

Our study detected an ll-deoxycortisol binding moiety in tissues that have been
characterized for glucocorticoid receptor in salmonids (Chakraborti et al. 1987;
Chakraborti and Weisbart 1987; Pottinger 1990). In addition, speciﬁc ll-deoxycortisol
binding moiety was observed in all tissues tested, however, much higher capacity was
found in gill, intestine, testis and much lower in liver, kidney, heart and muscle.

Our study has shown that the sea lamprey ll-deoxycortisol receptor complex
binds to DNA-cellulose, indicating that it is a nuclear steroid receptor. Similar to earlier
glucocorticoid receptor studies, heat activation was important for increasing the levels of
speciﬁc binding during DNA-cellulose chromatography (Eisen and Glinsmann 1978;
Knoebl et al. 1996). This study showed glucocorticoid receptor complex eluting from the
DNA-cellulose with 0.4 M NaCl column buffer, which was similar to the ﬁndings of
Knoebl et al. (1996). No further speciﬁc binding or nonspeciﬁc binding peaks were
found after elution with the 2.0 M NaCl wash buffer, indicating that activated receptor
complex binds to DNA-cellulose.

Functional corticosteroid binding globulin (CBG) has not been found in lower
vertebrates (Breuner and Orchinik 2002). Breuner and Orchinick (2002) report that a
cDNA sequence of a CBG homologue was found in zebraﬁsh, however, the authors

doubted that it ﬁinctioned as a CBG. There is no other evidence supporting CBG in

57

lower vertebrates. Therefore, it is unlikely that the binding moiety is a CPG in our study.
The results demonstrated that the ll-deoxycortisol receptor complex binds to DNA.

Recent studies have focused on steroid receptors and their role in increasing
regulatory and developmental complexity in vertebrates (Baker 1997; Baker 2001; Baker
2004; Whitﬁeld et al. 1999). There is evidence that teleostean ﬁsh experienced an
additional whole-genome duplication event that led to increased speciation and
biodiversity (Hoegg et al. 2004; Volff 2005). Thornton (2001) provided evidence that the
ancient corticoid receptor gene may have experienced a gene duplication event, which led
to the development of glucocorticoid and mineralocorticoid receptors in later vertebrates.

A previous study found ll-deoxycorticosterone circulating in the ancestral
vertebrate (chapter 1) and that the steroid had a higher relative afﬁnity to the receptor
compared to other corticosteroids tested. Recently, Sturrn et al. (2005) using
transactivation assays provided evidence that 11-deoxycorticosterone might be the
ancestral hormone associated with the mineralocorticoid receptor in rainbow trout
Oncorhynchus mykiss. Gillrnour (2005) suggested that in order to establish that 11-
deoxycorticosterone as a mineralocorticoid receptor ligand, it must increase in circulation
with a physiological stressor. In addition, it should not only increase, but it must also be
used in characterization of the receptor and show biological effects. The recent ﬁndings
suggest that 1 1-deoxycorticosterone might be the putative hormone for the ancestral
mineralocorticoid receptor which arose after agnathans, but before chondrichthyes
diverged (Bury and Sturm 2007).

Our study provided the ﬁrst characterization of a glucocorticoid receptor in one of

the earliest extant vertebrates, the sea lamprey. The results provide evidence that 11-

58

deoxycortisol is highly speciﬁc for the glucocorticoid receptor. The next step in
conﬁrming that ll-deoxycortisol is the actual hormone is to provide evidence of

biological effects.

59

ACKNOWLEDGEMENTS

We thank the staff of the US. Geological Survey, Hammond Bay Biological
Station for supplying sea lampreys. I would like to thank the Bonneville Power
Administration for providing funding and support for this research. I would also like to

thank the Confederated Tribes of the Umatilla Indian Reservation for support.

60

REFERENCES

Baker, ME. 1997. Steroid receptor phylogeny and vertebrate origins. Molecular and
Cellular Endocrinology 135:101-107.

Baker, ME. 2001. Adrenal and sex steroid receptor evolution: environmental
implications. Journal of Molecular Endocrinology 26:119-125.

Baker, ME. 2004. Co-evolution of steroidogenic and steroid-inactivating enzymes and
adrenal and sex steroid receptors. Molecular and Cellular Endocrinology 215:55-62.

Bertrand, S., Brunet, F .G., Escriva, H., Parmentier, G., Laudet, V., and Robinson-
Rechavi, M. 2004. Evolutionary genomics of nuclear receptors: from twenty-ﬁve

ancestral genes to derived endocrine systems. Molecular Biology and Evolution
21:1923-1937.

Breuner, C.W., and Orchinik, M. 2002. Plasma binding proteins as mediators of
corticosteroid action in vertebrates. Journal of Endocrinology 175:99-112.

Bridgham, J .T., Carroll, S.M., and Thornton, J .W. 2006. Evolution of hormone-receptor
complexity by molecular exploitation. Science 312297-101.

Bury, NR, and Sturm A. 2007. Evolution of the corticosteroid receptor signaling
pathway in ﬁsh. General and Comparative Endocrinology 153247-56.

Chakraborti, PK, and Weisbart, M. 1986. High-afﬁnity cortisol receptor activity in the
liver of the brook trout, Salvelinusfontinalis (Mitchill). Canadian Journal of Zoology
65:2498-2503.

Chakraborti, P.K., Weisbart, M., and Chakraborti, A. 1987. The presence of
corticosteroid receptor activity in the gills of the brook trout, salvelinusfontinalis.
General and Comparative Endocrinology 66:323-332.

Eisen, H.J., and Glinsmann, W.H. 1978. Maximizing the puriﬁcation of the activated
glucocorticoid receptor by DNA-cellulose chromatography. Biochemical Journal
171:177-183.

Escriva, H., Saﬁ, R., Hanni, C., Langlois, M-C., Saumitou-Laprade, P., Stehelin, D.,
Capron, A., Pierce, R., and Laudet, V. 1997. Ligand binding was acquired during
evolution of nuclear receptors. Proceedings of the National Academy of Sciences
94:6803-6808.

61

Gillmour, KM. 2005. Mineralocorticoid receptors and hormones: ﬁshing for answers.
Endocrinology 146:44-46.

Ho, S-M., Press, D., Liang, L-C., and Sower, S. 1987. Identiﬁcation of an estrogen
receptor in the testis of the sea lamprey, Petromyzon marinas. General and
Comparative Endocrinology 67:119-125.

Keay, J ., Bridgham, J .T., and Thornton, J .W. 2007. The octopus vulgaris estrogen
receptor is a constitutive transcriptional activator: evolutionary and functional
implications. Endocrinology 147 :3861-3869.

Knoebl, I., Fitzpatrick, MS, and Schreck, CB. 1996. Characterization of a
glucocorticoid receptor in the brains of Chinook sahnon, oncorhynchus tshawytscha.
General and Comparative Endocrinology 101:195-204.

Kumar, S., and Hedges, SB. 1998. A molecular timescale for vertebrate evolution.
Nature 392:917-920.

Laudet, V. 1997. Evolution of the nuclear receptor superfamily: early diversiﬁcation from
an ancestral orphan receptor. Journal of Molecular Endocrinology 19:207-226.

Maule, A.G., and Schreck, CB. 1991. Stress and cortisol treatment changed afﬁnity and
number of glucocorticoid receptors in leukocytes and gill of coho salmon. General
and Comparative Endocrinology 84:83-93.

Patino, R., and Thomas, P. 1990. Characterization of membrane receptor activity for 17
a, 203, 21-trihydroxy-4-pregnen-3-one in ovaries of spotted seatrout (Cynoscion
nebulosus). General and Comparative Endocrinology 78:204-217.

Pottinger, TC. 1990. The effect of stress and exogenous cortisol on receptor-like binding
of cortisol in the liver of rainbow trout, Oncorhynchus mykiss. General and
Comparative Endocrinology 78: 194-203.

Pottinger, T.G., and Brierley, I. 1997 . A putative cortisol receptor in the rainbow trout
erythrocyte: stress prevents starvation-induced increases in speciﬁc binding of
cortisol. The Journal of Experimental Biology 200:2035-2043.

Robinson-Rechavi, M., Garcia, HE, and Laudet, V. 2003. The nuclear receptor
superfamily. Journal of Cell Science 116:585-586.

Shu, D-G., Luo, H-L., Monis, S.C., Zhang, X-L., Hu, S-X., Chen, L., Han, J., Zhu, M.,

Li, Y., and Chen, L-Z. 1999. Lower Cambrian vertebrates from south China. Nature
402242-46.

62

Sturm, A., Bury, N., Dengreville, L., Fagart, J ., Flouriot, G., Rafestin-Oblin, ME, and
Prunet, P. 2005. ll-Deoxycorticosterone is a potent agonist of the rainbow trout
(Oncorhynchus mykiss) mineralocorticoid receptor. Endocrinology 146:47-55.

Thornton, J .W. 2001. Evolution of vertebrate steroid receptors from an ancestral estrogen
receptor by ligand exploitation and serial genome expansions. Proceedings of the
National Academy of Sciences 98:5671-5676.

Thornton, J .W. 2003a. Nonmammalian nuclear receptors: evolution and endocrine
disruption. Pure Appl. Chem. 75: 1827-1839.

Thornton, J .W., Need, E., and Crews, D. 2003. Resurrecting the ancestral steroid
receptor: ancient origin of estrogen signaling. Science 301 :1714-1717.

Volff, J -N. 2005. Genome evolution and biodiversity in teleost ﬁsh. Heredity 94:280-294.
Whitﬁeld, G.K., Jurutka, P.W., Haussler, CA., and Haussler, MR. 1999. Steroid

hormone receptors: evolution, ligands, and molecular basis of biologic ﬁrnction.
Journal of Cellular Biochemistry Supplements 32/33:110-122.

63

CHAPTER 3

BIOLOGICAL ROLES OF ll-DEOXYCORTISOL IN MEDIATING STRESS

RESPONSES OF THE SEA LAMPREY Petromyzon marinus

64

ABSTRACT

This study was conducted to assess whether ll-deoxycortisol serves as a
glucocorticoid in the ancient vertebrate, the sea lamprey Petromyzon marinas. Earlier
research has shown that 1 1-deoxycortisol circulates in the blood and has a speciﬁc
receptor in the gills of the sea lamprey. In this study, the hypothesis was that 11-
deoxycortisol would exhibit a classical stress response and have biological effects. After
acute stress, an increase in ll-deoxycortisol levels occurred by 1 h and remained elevated
for at least 8 h, recovering to basal level by 24 h. 1 1-deoxycortisol implants elevated
circulating levels of 1 1-deoxycortisol and reduced levels of classic androgens and
estrogens in the plasma. The implants also increased gill Na+, K+-ATPase activity in
lamprey. Mammalian corticotropin releasing hormone injections sharply increased
plasma levels of 1 l-deoxycortisol. In addition, lamprey pituitary extract injections
increased plasma concentrations of 1 l-deoxycortisol in a dose response manner. This
study provided strong evidence that 11-deoxycortisol is an ancient glucocorticoid in the

lamprey.

65

INTRODUCTION

The stress response is vital to vertebrates and is well conserved among the jawed
vertebrates. In tetrapods, an important component of the stress steroid response system is
the hypothalamic-pituitary—adrenal (HPA) axis (Chrousos 1998). Teleosts have the same
system, except they secrete corticosteroids from interrenal cells located in the anterior
portion of the kidneys (N andi and Bern 1960; Wendelaar Bonga 1997; Barton 2002).
The secretion of the corticosteroids is mainly under the control of the neuropeptides
corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH)

(W endelaar Bonga 1997; Maj zoub 2006). The hypothalamus is stimulated to release
CRH, which in turn mediates the release of ACTH from the pituitary in jawed
vertebrates. ACTH then binds to receptors in the adrenal or interrenal cells to release
corticosteroids into circulation. While the physiological stress steroid response is well
conserved in vertebrates, the chemical structures of the corticosteroids are different.

There are two types of corticosteroids in vertebrates, the glucorticoids and
mineralocorticoids. Glucocorticoids regulate metabolism and are released in response to
a stressor (Sapolsky et al. 2000). Cortisol and corticosterone, which differ only by the
absence a 17-01 hydroxyl, are the two principal glucocorticoids in animals. Mammals
secrete cortisol; while rats and mice secrete corticosterone. Reptiles and birds also

produce and circulate corticosterone as their primary glucocorticoid (Nelson 2000). Fish,

66

on the other hand, secrete cortisol as their glucocorticoid (Idler and Truscott 1972;
Mommsen et al. 1999; Wendelaar Bonga 1997; Barton 2002), with the exception of
elasmobranchs which synthesize la—hydroxycorticosterone (Idler and Truscott 1967;
Nunez and Trant 1999). It was previously thought that glucocorticoids in ﬁsh also
functioned as mineralocorticoids (Wendelaar Bonga 1997).

Aldosterone is the principal mineralocorticoid in terrestrial vertebrates (Nelson
2000). Aldosterone is important in regulation of mineral and ion balance in vertebrates
(Norman and Litwack 1997). Evidence to support aldosterone circulating in ﬁsh seems
to be absent (Gilmour 2005; Bury and Sturm 2007). However, Sturm et al. (2005) has
shown that ll-deoxycorticosterone is speciﬁc for a cloned portion of the
mineralocorticoid receptor in rainbow trout. Their study suggests that 11-
deoxycorticosterone may be a putative mineralocorticoid in ﬁsh (Sturm et al. 2005).
Bridgham et al. (2006) found a mineralocorticoid receptor homologue in the skate, Raja
erz’nacea but not in lamprey.

It was unclear if one of the earliest vertebrates, the lamprey, contains a stress
steroid. The lamprey is an agnathan, one of the oldest extant vertebrates which emerged
over 500 million years ago (Kumar and Hedges 1998; Shu et al. 1999). To discover the
actual stress hormone would be an important step in understanding the evolution of
steroid receptor systems in the vertebrates.

Two putative glucocorticoids were identiﬁed, ll-deoxycortisol and 11-
deoxycorticosterone, circulating in the lamprey plasma (Chapter 1). Further, the results
from chapter 2 have shown that ll-deoxycortisol has a speciﬁc glucocorticoid receptor in

the gill of lamprey. The last lines of evidence needed to support the claim that 11-

67

deoxycortisol is an ancestral glucocorticoid was to show classic glucocorticoid response
to stress and to show biological effects. The speciﬁc questions for this study are: 1) Does
ll-deoxycortisol show a classical stress steroid response, sharply rising and returning to
basal levels? 2) Will ll-deoxycortisol implants reduce classical androgens and estrogens
in circulation and increase gill Na+, K+-ATPase activity? 3) Will mammalian CRH
increase circulating levels of 1 l-deoxycortisol? 4) Will lamprey pituitary extract increase

circulating levels of 11-deoxycortisol?

68

MATERIALS AND METHODS

Source of Animals

Sea lamprey, Petromyzon marinus, were collected in streams by US. Fish and
Wildlife Service employees, and transported to Michigan State University (East Lansing,
M1) or Hammond Bay Biological Station (Millersburg, MI) for collecting tissues where
they were held at 10-13 °C. Tissues collected from ﬁsh were frozen in liquid nitrogen
and held at -80 °C until processed. All experiments were approved by the Michigan State

University Institutional Animal Care and Use Committee (AUF # 05/04-077-00).

Handling and Salinity Stressors

Adult lampreys were acclimated in ﬂow through tanks (254 L) at least 2 weeks
before stress tests were conducted. Tanks were isolated to keep people from disturbing
the lampreys during acclimation. In the ﬁrst stress experiment (1-48 h recovery), 140
lampreys were distributed in tanks at a density of 7 lampreys/tank with replicate tanks for
each treatment at each time. No lampreys were sampled more than once. In the second
stress experiment (1-24 h recovery), 80 parasitic lampreys were distributed in tanks at a

density of 5 lampreys/tank with replicate tanks for each treatment at each time. No

69

lampreys were sampled more than once. Lampreys were netted out of tanks and placed
in a dry bucket for 5 min then transferred to 3 % salt water for 10 min. Handling and
salinity tests are routine methods to elicit a stress steroid response in teleosts
(Wedemeyer et al. 1990). To obtain plasma, lampreys were netted out of tanks and
immersed in anesthetic dose of 400 mg/L of tricaine methanesulfonate (MS-222)
buffered with sodium bicarbonate. Blood samples were collected at 1, 4, 8, 24, and 48 h
after stressors to measure steroid levels. For parasitic lampreys, blood was sampled at 1
and 24 h after stressors. Lampreys were then euthanized with a lethal dose of MS-222.
Blood was centrifuged at 1000 x g at 4°C for 15 min and plasma removed. Plasma was

stored at -80 °C until analysis.

Radioimmunoassay (RIA) procedure

RIAs were conducted in glass culture tubes (10 mm X 75mm, Fisher Scientiﬁc,
Pittsburgh, PA). Brieﬂy, the assay buffer consisted of 50 mM sodium phosphate, pH 7.4,
0.2% BSA, 137 mM NaCl, 0.40 mM EDTA, and 0.77 mM sodium azide. Nine standards
were made up in duplicate over the range 500-1.95 pg/ 100 pl/tube. The tubes containing
unknowns also had a volume of 100 ul. Binding reagent was made by adding radiolabel
and antiserum to 20 ml of assay buffer in amounts such that, when 100 ul was dispensed
to all tubes, each tube contained 5000 dpm and in the absence of any standard, 50% of
the radiolabel was bound to the antiserum. Blank tubes, to which no antibody was added,
and tubes necessary to determine the total and maximum dpm counts, were also included

in the assay. All tubes were incubated overnight at 4 °C. After overnight incubation, 500

70

pl of ice-cold charcoal solution (50 mM sodium phosphate, Pb 7.4, 0.1% gelatin, 1.0%
dextran-coated charcoal) was added to each tube. The tubes were kept on ice for 20 min,
and then centrifuged at 1000 x g for 12 min. The supematants were poured into 8 ml
scintillation vials, mixed with 6 ml scintillation ﬂuid and DPM were counted using a

scintillation counter.

Steroid implants

11-Deoxycortisol (Sigma-Aldrich, St. Louis, MO) time release pellets were made
by Innovative Research of America (Sarasota, F1). The 21-day slow release steroid
implants (5 mg/pellet) were injected between the muscle and the skin near the front
dorsal ﬁn of the sea lamprey. A total of 48 lampreys were distributed in ﬂow through
tanks (254 L) at a density of 6 lampreys/tank with replicate tanks for each treatment. On
the 21St day, blood samples and gill tissues were collected. Plasma was analyzed by RIAs
for ll-deoxycortisol (S), 1 1-deoxycorticosterone (DOC), dehydroepiandrosterone-sulfate
(DHEA-S), dehydroepiandrosterone (DHEA), androstenedione (AD), testosterone (T),

estrone (E1), and estradiol (E2).

Gill Na+, K+-ATPase Activity

A gill pouch was removed and placed in ice-cold SEI buffer (150 mM sucrose, 10

mM EDTA, 50 mM imidazole, pH 7.3) and frozen immediately at -80 °C. Na+, K'-

ATPase activity was determined with a kinetic assay run in 96-well microplates at 25 °C

71

and read at a wavelength of 340 nm for 10 min as described in McCormick (1993). Gill
tissue was homogenized in 500 111 SEID (SEI buffer and 0.1% deoxycholic acid) and
centrifuged at 5000 x g for 30 5. Ten pl samples were run in two sets of duplicates; one
set containing the assay mixture and the other assay mixture and 0.5 mM ouabain. The
resulting ouabain-sensitive ATPase activity is expressed as pmoles ADP/mg protein/h.
Protein concentrations were determined using BCA (bicinchoninic acid) Protein Assay
(Pierce, Rockford, 11). Both assays were run on a THERMOmax microplate reader using

SOFTmax software (Molecular Devices, Menlo Park, CA).

Corticotropin Releasing Hormone Injections

Mammalian corticotropin releasing hormone (CRH) (Si gma-Aldrich) was
dissolved in 0.9% saline and injected intraperitoneally with a dose of 100 pg/Kg. Saline
solution was used as a control. Blood samples were collected 1 hr after injections by
cardiac puncture using vacutainers (Becton Dickinson, Franklin Lakes, NJ ,). Blood
samples were centrifuged at 1000 x g for 15 min; plasma was collected and stored at -80

°C until analyzed by RIA for S.

Pituitary extract injections

To obtain pituitary extract, sea lamprey pituitary glands from 400 adults were

collected in June 2000, at Hammond Bay Biological Station (Millersburg, MI, USA).

The frozen pituitary glands were homogenized in 20 ml of 20-mM Tris buffer, pH 7,

72

containing protease inhibitor cocktails (Roche, Nutley, NJ, USA). This mixture was
centrifuged at 1000g for 20 min allowing recovery of the supernatant. The protein
concentration was determined using DCA protein analysis kit (Pierce, Rockford, IL).
The protein concentration for the extract was 6.7 mg/ml. One milliliter of the extract was
equivalent to 201amprey pituitary glands. Lampreys were given a single intraperitoneal
injection of equivalent to 1, 5, or 10 pituitaries or a 0.9% saline as a control (4 treatments
total, 10 lampreys per treatment). Blood was sampled at 0, 6, 12, 24, and 48 h after the
injection. Blood samples were centrifuged at 1000 x g for 15 min; plasma was collected

and stored at -80 °C until analyzed by RIA for S.

Statistical analysis

Statistical analysis of the acute stress experiments was done using Analysis of
Variance (AN OVA) in which time was an independent variable. Comparisons among
time intervals were compared using Bonferroni’s multiple comparison tests. Analysis of
the implant experiments was done using an Unpaired t test with Welch’s correction.
Males and females were analyzed separately. The corticotropin releasing hormone
injection experiment was also analyzed by an unpaired t test. Analysis of the pituitary
extract experiment was done with repeated measures two-way ANOVA in which

pituitary equivalent dosage and time were factors.

73

RESULTS

Acute Stress Response

Spawning phase adult lampreys subjected to a 5-min dewatering and 3% salt
water bath (10 min) exhibited a sharp increase in S levels within 1 hr (Fig 1A). Plasma S
levels in control lampreys were 1.09 i 0.06 ng ml”, while stressed lampreys elevated
levels to 2.38 :t 0.14 pg ml'1 (P < 0.001) at l h. Concentrations of plasma S remained
elevated at least for 8 h, returning to control levels by 24 h.

Parasitic male and female lampreys subjected to a 5-min dewatering and 3% salt
water bath (10 min) exhibited a sharp increase in S levels within 1 hr (Fig. 1B). Plasma S
levels in stressed male lampreys was 1.08 :l: 0.09 ng ml", compared to control levels 0.45
i 0.04 ng ml'l (P < 0.001) at 1 h. Plasma S levels in stressed female lampreys was 1.46 :l:
0.19 ng ml”, compared to the control levels 0.39 :l: 0.01 ng ml'1 (P < 0.001) at 1 h.
Plasma levels of S in male and female parasitic phase lampreys returned to control levels
by 24 h. We found basal levels of S nearly doubled from the parasitic stage to the

spawning phase in lampreys.

Effects of steroid implants

The S implants caused an increase in plasma levels of S and DOC in male and

female lampreys (Fig. 2). S implanted male lampreys had a mean S level of 18.9 :1: 2.0 ng

ml”, compared to 1.1 i .06 ng ml'1 (P < 0.0001) in control implanted lampreys (Fig. 2A).

74

Implanted female lampreys had a mean S level of 20.5 d: 3.0 ng ml’l , compared to 1.2 :l:
0.09 ng ml'1 (P < 0.0001) in control lampreys. The S implants also increased circulating
DOC in males from 0.84 i 0.02 ng ml" to 2.55 :h 0.14 ng ml" (P < 0.0001) (Fig. 2B).
The S implants increased circulating DOC levels from 0.54 :l: 0.02 ng ml" to 1.95 :l: 0.17
ng ml'1 (P < 0.0001) in female lampreys.

The S implants caused a decrease in plasma levels of DHEA-S and DHEA in
male and female lampreys (Fig. 3). Implanted male and female lampreys decreased mean
plasma DHEA-S levels from 2.66 a: 0.15 ng ml" and 1.61 i 0.07 ng ml" to 2.16 i 0.11 ng
ml'1 and 1.30 :I: 0.07 ng ml" (P = 0.01) respectively (Fig. 3A). Implanted male and
female lampreys decreased mean plasma DHEA levels from 0.32 i 0.01 ng ml'1 and 0.28
a: 0.008 ng ml" to 0.29 i: 0.008 mg ml" (P = 0.04) and 0.21 :t 0.02 ng ml" (P = 0.02)
respectively (Fig. 3B).

The mean plasma levels of AD in male and female lampreys implanted with S
were not signiﬁcantly different (Fig. 4A). However, plasma levels of T decreased in
male and female lampreys implanted with S (Fig. 4B). Implanted male and female T
levels decreased from 0.31 :1: 0.005 ng ml'1 and 0.23 i 0.01 ng ml" to 0.27 i 0.01 ng ml'1
(P = 0.002) and 0.17 1. 0.01 ng ml" (P = 0.005) respectively.

Implants caused a decrease in mean plasma level of E1 in male lampreys, but not
in female lampreys (Fig. 5A). Mean plasma E1 levels in males implanted with S was
1.76 i 0.15 ng ml", while plasma levels in control lampreys were 2.46 i 0.18 ng ml'1 (P
= 0.009). lrnplants decreased plasma levels of E2 in male and female lampreys (Fig. 5B).

The mean plasma level of E2 in control males and females was 3.50 i 0.28 ng ml'1 and

75

2.03 :1: 0.13 ng ml", with levels decreasing to 2.44 :l: 0.21 ng ml" (P = 0.008) and 1.12 i

0.12 ng ml'1 (P = 0.0001) respectively.

Na+, K+-ATPase Activity

The S implants nearly doubled gill Na+, K+-ATPase activity in both male and
female lamprey after 21 days (Fig. 6). The gill Na+, K+-ATPase activity was 2.9 d: 0.23
pmol ADP/mg protein/h and 2.8 :t 0.22 pmol ADP/mg protein/h, in control male and
female lamprey, with levels increasing to 5.7 :l: 0.53 pmol ADP/mg protein/h (P <

0.0001) and 4.1 i 0.30 pmol ADP/mg protein/h (P = 0.002).
Corticotropin Releasing Hormone Injections

Mammalian CRH injections markedly increased plasma S levels in male and
female lampreys 1 hr after injection (Fig. 7). Mean plasma S level in saline injected male
lampreys was 0.99 i 0.13 ng ml" and increased to 2.08 i: 0.39 ng ml" (P = 0.02). Mean
plasma S level in saline injected female lampreys was 0.72 :l: 0.12 ng ml'1 and increased
to 2.84 i 0.28 ng ml" (P = 0.001).

Pituitary Extract Injections

Pituitary extract injections increased plasma S levels in a dose dependent manner

(Fig. 8). The 1, 5, and 10 pituitary equivalent treatments signiﬁcantly increased plasma S

76

levels to 2.11 d: 0.51 (P = 0.002), 3.16 i: 0.55 ng ml'1 (P < 0.0001), and 3.31 :t 0.52 ng ml'
1 (P < 0.0001) from a control level of 1.35 :l: 0.40 ng ml'1 at 6 h. By 12 h, all treatment
groups except for the 10 pituitary equivalent group returned to control levels. At 48 h,

the l pituitary equivalent group was slightly reduced compared to control levels.

77

2'5 i I 1: Control
** ** I: Stress

 

 

 

 

 

 

 

 

 

 

2.0 - i I}
E 1.5 -
a .
5 1.0-
g .
E 0.5
8 0 0 '
>1
3 1 4 8 24 48
w n
a. 2 o . B Time (h)
‘—
To ' ** :1 1h Control
E 1.5 . _ 1h Stress
to m 24h Control
2 ** 1:! 24 h Stress
n.

1.0 -

0.5 -

0.0 ,

Male Female

Figure 3-1. Effect of handling and salt challenge stressors on plasma levels of 11-
deoxycortisol for spawning (A) and parasitic (B) phase lamprey during recovery.
Vertical bars represent means i SEM of 6-14 observations. *, P < 0.05; **, P < 0.01;
***, P < 0.001.

***

31’
>

1+.

1: Control
I: S implant

.3
UI

(nglml)

10:

Plasma 11-Deoxycortisol

 

 

 

 

 

3.0 - B m

2.5 -' "Ii
2.0 -' 3f

1.5 -
1.0 -

.°
01

 

 

 

 

 

0.0 - i H- '
Male Female

 

Plasma 11-Deoxycorticosterone
(nglml)

Figure 3-2. Effect of 21 d ll-deoxycortisol implant treatment on plasma levels of 11-
deoxycortisol (A) and ll-deoxycorticosterone (B). Vertical bars represent means :1: SEM
of 1 1-12 observations. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

79

9"
o

:1 Control
S implant

lilil>
1+.
1+1

_L
O

Plasma DHEA-S (nglml)

 

 

 

 

 

 

 

 

 

99
#0

0.3- {-2 {E-
. *

0.1 1

Plasma DHEA (nglml)
O
N
I-H

 

 

 

 

 

 

 

 

 

 

0.0 . " . ‘
Mal Female

Figure 3-3. Effect of 21 d ll-deoxycortisol implant treatment on plasma levels of
Dehydroepiandrosterone-sulfate, DHEA-S (A) and Dehydroepiandrosterone, DHEA (B).
Vertical bars represent means i SEM of 11-12 observations. *, P < 0.05; **, P < 0.01;
***, P < 0.001.

80

 

 

d)
g . A 1:3 Control
:5 [:1 S implant
o 0.3 -
E, 2
*2 s
.5 g 0.2 -
c I
<
E 0.1 -
1n
.9 1
m
0.0 .
0.4 -

 

 

 

 

 

 

 

 

 

a) .
c
g 0.3- T" **
1;; c 1*
*3 E 02
.2 .3.“
g .
m 0.1 ~
2
Q-

0.0

Male Female

Figure 3-4. Effect of 21 d 1 1-deoxycortisol implant treatment on plasma levels of
androstenedione (A) and testosterone (B). Vertical bars represent means i SEM of 11-12
observations. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

81

3.0 -

2.5 -

1.5 -
1.0 .

0.5 '

Plasma Estrone (nglml)

0.0

 

 

 

 

[:1 Control
CI] 3 implant

 

 

4.0 -

3.0 -

2.0 -

1.0 -

 

Plasma Estradiol (nglml)

 

 

 

 

***

    

v - unmuu

 

0.0

Male

Female

Figure 3-5. Effect of 21 d 11-deoxycortisol implant treatment on plasma levels of estrone
(A) and estradiol (B). Vertical bars represent means i SEM of 11-12 observations. *, P
< 0.05; **, P < 0.01; ***, P < 0.001.

82

***

:13

Nw-hOION

Na"'-K"'-ATPase Activity
(pmol ADP/mg protein/h)

 

 

 

lulu“ llllll

:1 Control
I: S implant

**

{—

 

 

 

0

Female

Figure 3-6. Effect of 21 d ll-deoxycortisol implant treatment on gill Na: K+-ATPase
activity. Vertical bars represent means :1: SEM of 11-12 observations. *, P < 0.05; **, P

< 0.01; ***, P < 0.001.

83

 

 

 

 

 

 

g 35 . 1:1 Saline injected

2) :1 CRH injected **
: 3.0 - _E_
o

.2 2.5- *

E

o 2.0 -

2

o 1.5 -

8

‘; 1.0 -

‘_

«1 0.5 -

E

g 0.0 . -

0- Male Female

Figure 3-7. Changes in concentration of plasma 1 1-deoxycortisol following injections of
mammalian CRH. Vertical bars represent means i SEM of 3-5 observations. *, P <
0.05; **, P < 0.01; ***, P < 0.001.

84

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

E l:|Saline(n=9)

El 4000' i i I:I1x(n=10)

E'- * -5x(n=9)

3 -10x(n=10)

53 3000- *

E * *

0

5‘ 2000- j

3 l l *

, 1 ~ 1 11 l l

S 1000-

to

E

8 0J__-_ _.lh. Li. —-H L..—

E 0 6 12 24 48
Time (h)

Figure 3-8. Changes in concentration of plasma ll-deoxycortisol following injections of
pituitary extract. Vertical bars represent means i SEM of 9-10 observations. *, P < 0.05;
**, P < 0.01; ***, P < 0.001.

85

DISCUSSION

Our study has provided several lines of evidence to support the hypothesis that S
is an ancestral glucocorticoid hormone. The results demonstrated that circulating S
responds to acute stress in the ancient vertebrate, the sea lamprey. In lamprey, S exhibits
similar characteristics to the classical cortisol responses in teleosts. In this study, S levels
increased signiﬁcantly within 1 hour of an acute stressor, remained elevated for at least 8
h, and returned to basal levels by 24 h. In most teleosts, cortisol levels generally increase
rapidly, peaking within 1-2 h after exposure to an acute stressor, remain elevated,
followed by recovery (Pickering et al. 1987 ; Barton 2002; Milston et al. 2006). Plasma S
levels doubled after acute stress in lamprey, however, the levels were found to be lower
than cortisol levels measured in most ﬁshes (Barton and Iwama 1991; Barton 2002).

This study showed that S response to acute stress in lamprey was most similar to cortisol
levels in stressed Pallid sturgeon Scaphirhynchus albus (Barton 2002). Parasitic phase
lamprey had lower basal plasma S levels compared to spawning phase lamprey. The
pattern of increasing cortisol levels during spawning phase salmonids is well documented
(Carruth et al. 2000).

Our study demonstrated that S has classic glucocorticoid effects such as
decreasing androgens, estrogens, and increasing gill Na+, K+-ATPase activity in the
ancient vertebrate. This study found that S implants decreased classical androgens in the
plasma of lamprey. The S implants also signiﬁcantly reduced plasma levels of androgens
DHEA-S, DHEA and T. The synthetic glucocorticoid dexamethasone has been shown to

decrease DHEA-S, DHEA, AD, and T in humans (Abraham 1974; Kalimi et al. 1994).

86

Acute and chronic stressors have been shown to elevate plasma glucocorticoid, while
decreasing T in ﬁsh (Pickering et al. 1987; Pankhurst and Dedual 1994), amphibians
(Moore and Zoeller 1985), and reptiles (Lance and Elsey 1986). Cortisol implants were
also shown to be effective at reducing plasma levels of T in ﬁsh (Carragher et al. 1989;
F00 and Lam 1993A,B). Interestingly, S implants did not reduce circulating AD in
lamprey, even though Bryan et al. (2007) provided evidence that AD was an androgen in
the sea lamprey. One possibility is that AD is not the principal androgen and that the
ancestral vertebrate utilizes the 5-ene pathway to T. It is well known that the biosynthetic
pathway can go from DHEA to androstenediol then to T (Grower 1995).

Our study demonstrated that plasma levels of E2 in both sexes and E1 in males
signiﬁcantly decrease after S implants in lamprey. Similar to the current ﬁndings, acute
stress and cortisol implants were shown to decrease plasma levels of E2 in teleosts
(Pankhurst and Dedual 1994; F00 and Lam 1993A). E2 has been well studied in lamprey
and established as a sex hormone in lamprey. Ho et al. (1987) provided evidence for an
E2 receptor in the testis of the sea lamprey. Plasma levels of E2 were shown to increase
during the reproductive stage in lamprey (Bolduc and Sower 1992) and increased with
injections of lamprey GnRH I, and III (Deragon and Sower 1994; Gazourian et al. 1997).
In addition, E2 was shown to stimulate vitellogenesis in female and male lampreys
(Mewes et a1. 2002).

The S implants increased gill Na+, K+-ATPase activity in adult lamprey. Our
results show that S implants signiﬁcantly increased ion regulating proteins in the gills of
male and female lampreys. In juvenile sahnonids, cortisol and growth hormone have

been shown to increase gill Na+, K+-ATPase activity (McCormick 1996). Cortisol

87

administration by itself has also been shown to increase gill Na+, K+-ATPase activity
(Madsen 1990; McCormick et al. 1991; McCormick 1996; Seidelin et al. 1999; Quinn et
al. 2003) and intestinal Na+, K+-ATPase activity (Veillette and Young 2005) in juvenile
salmonids.

Our results have demonstrated that S is regulated by the hypothalamus-pituitary
axis in the ancient vertebrate. The mammalian CRH injections increased plasma levels of
S in male and female sea lampreys. CRH elicits a pronounced increase in plasma S
compared to the saline injected lampreys. In teleosts, the regulation of cortisol release is
under the hypothalamus-pituitary-interrenal axis (Mommsen et al. 1999). CRH
neuropeptide controls the release of ACTH from the anterior pituitary which circulates in
the blood and binds to receptors in the interrenal cells, which in turn, stimulates release of
cortisol (Wendelaar Bonga 1997). In goldﬁsh, injections of CRH, urotensin I, and
sauvagine elicited signiﬁcant increases in plasma cortisol and stimulated release of
ACTH from interrenals (Fryer et al. 1983). Pepels et al. (2004) showed signiﬁcant
increases of plasma levels of CRH and cortisol by 5 min in tilapia. The CRH peptide
sequences in the vertebrates are highly conserved (Lovejoy and Balrnent 1999). Lovejoy
and J ahan (2006) suggest the CRH system based on CRH homologues found in insects
and chordates may have evolved in a metazoan ancestor.

Pituitary extract injections increased plasma levels of S in a dose-response
manner. The increases in plasma S from control levels were substantially elevated at 6 h
for all treatment groups. All treatment groups recovered to control levels by 24 h.

ACTH secreted from the pituitary is the principal hormone that controls cortisol secretion

in teleosts (Wendelaar Bonga 1997). Pickering et al. (1987) have shown increased levels

88

of plasma ACTH in brown trout after acute stress. In lamprey, ACTH has been cloned
and sequenced from the pituitary (Heinig et al. 1995; Takahashi et al. 1995A; Takahashi
et al. 1995B). The lamprey ACTH is 60 amino acids long, roughly 20 amino acids
longer than other vertebrate ACTHs (Takahashi et a1. 2006). However, plasma ACTH
levels have not been shown to increase in lamprey and direct stimulation of 11-
deoxycortisol by lamprey ACTH has not been shown.

In teleosts, there is evidence of other pituitary hormones producing corticotrOpic
activity (Wendelaar Bonga 1997; Mommen et al. 1999). Schreck et a1. (1989)
demonstrated that gonadotropin hormone stimulated higher levels of cortisol release than
ACTH in coho sahnon Oncorhynchus kisutch interrenals. Recently, Sower et al. (2006)
cloned and sequenced a gonadotropin like protein from the pituitary of sea lamprey.
Therefore the stimulatory effect from the pituitary injection might not be lamprey ACTH,
but instead another factor or peptide hormone causing the increase in plasma S levels.

In conclusion, this study has demonstrated that S functions as a glucocorticoid
and has biological effects in the ancestral vertebrate, the lamprey. The results have
shown that S, like cortisol in higher vertebrates, exhibits a classical stress steroid
response in lamprey. In addition, the glucocorticoid hormone S reduced circulating
levels of classic androgens and estrogens in the plasma of lamprey. The glucocorticoid
hormone S, like cortisol in teleosts, up regulates ion regulating proteins in the gills of sea
lamprey. Lastly, we provide evidence that the lamprey glucocorticoid hormone S is

controlled by the hypothalamus-pituitary axis in these ancient vertebrates.

89

ACKNOWLEDGEMENTS

We thank the staff of the US. Geological Survey, Hammond Bay Biological
Station for supplying sea lampreys. I would like to thank the Bonneville Power
Administration for providing funding and support for this research. I would also like to

thank the Confederated Tribes of the Umatilla Indian Reservation for support.

90

REFERENCES

Abraham, GE. 1974. Ovarian and adrenal contribution to peripheral androgens during
the menstrual cycle. Journal of Clinical Endocrinology and Metabolism 39:340-346.

Barton, BA. 2002. Stress in Fishes: A diversity of responses with particular reference to
changes in circulating corticosteroids. Integrative and Comparative Biology 42:517-
525.

Barton, B.A., and Iwama, GK. 1991. Physiological changes in ﬁsh from stress in
aquaculture with emphasis on the response and effects of corticosteroids. Annual
Review of Fish Diseases 1:3-26.

Bolduc, T.G., and Sower, SA. 1992. Changes in brain gonadotropin-releasing hormone,
plasma estradiol 17-0, and progesterone during the ﬁnal reproductive cycle of the
female sea lamprey, Petromyzon marinas. The Journal of Experimental Zoology
264255-63.

Bridgham, J .T., Carroll, S.M., and Thornton, J .W. 2006. Evolution of hormone-receptor
complexity by molecular exploitation. Science 312:97-101.

Bryan, M.B., Scott, AP, and Li, W. 2007. The sea lamprey (Petromyzon marinus) has a
receptor for androstenedione. Biology of Reproduction 77:688-696.

Bury, NR, and Sturm, A. 2007. Evolution of the corticosteroid receptor signaling
pathway in ﬁsh. General and Comparative Endocrinology 153247-56.

Carragher, J .F ., Sumpter, J .P., Pottinger, T.G., and Pickering, AD. 1989. The deleterious
effects of cortisol implantation on reproductive ﬁrnction in two species of trout,
Salmo trutta L. and Salmo gairdneri Richardson. General and Comparative

Endocrinology 76:310-321 .

Carruth, L.L., Dores, R.M., Maldonado, T.A., Norris, D.O., Ruth, T., and Jones, RE.
2000. Elevation of plasma cortisol during the spawning migration of landlocked

kokanee sahnon (Oncorhynchus nerka kennerlyi). Comparative Biochemistry and
Physiology Part C 127:123-131.

Chrousos, GP. 1998. Stressors, stress, and neuroendocrine integration of the adaptive
response. Annals New York Academy of Sciences 851 :31 1-335.

Deragon, KL, and Sower, SA. 1994. Effects of lamprey gonadotropin-releasing
horrnone-III on steroidogenesis and sperrniation in male sea lampreys. General and
Comparative Endocrinology 95:363-367.

91

F 00, J .T.W., and Lam, T.J. 1993. Retardation of ovarian growth and depression of serum
steroid levels in the tilapia, Oreochromis mossambicus, by cortisol implantation.
Aquaculture 115:133-143.

Foo, J .T.W., and Lam, T.J. 1993. Serum cortisol response to handling stress and the
effect of cortisol implantation on testosterone level in the tilapia, Oreochromis
mossambz‘cus. Aquaculture 1 15: 145-1 58.

Fryer, J ., Lederis, K., and Rivier, J. 1983. Urotensin I, a CRF-like neuropeptide,
stimulates ACTH release from the teleost pituitary. Endocrinology 113:2308-2310.

Gazourian, L., Deragon, K.L., Chase, C.F., Pati, D., Habibi, HR, and Sower, SA. 1997.
Characteristics of GnRH binding in the gonads of effects of lamprey GnRH-I and —III
on reproduction in the adult sea lamprey. General and Comparative Endocrinology
108:327-339.

Gilrnour, KM. 2005. Mineralocorticoid receptors and hormones: Fishing for answers.
Endocrinology 146:44-46.

Grower, DB. 1995. Extraction, puriﬁcation and estimation of the androgens and their
derivatives. Pages 268-368 in H.L.J. Makin, D.B. Grower and D.N. Kirk, editors.
Steroid Analysis. Chapman & Hall, London.

Ho, S-M., Press, D., Liang, L-C., and Sower, S. 1987. Identiﬁcation of an estrogen
receptor in the testis of the sea lamprey, Petromyzon marinus. General and
Comparative Endocrinology 67:119-125.

Heinig, J .A., Keeley, F.W., Robson, P., Sower, S.A., and Youson, J .H. 1995. The
appearance of pr00piomelanocortin early in vertebrate evolution: cloning and
sequencing of POMC from a lamprey pituitary Cdna library. General and
Comparative Endocrinology 99:137-144.

Idler, DR, and Truscott, B. 1967. la-Hydroxycorticosterone: synthesis in vitro and
properties of an interrenal steroid in the blood of cartilaginous ﬁsh (Genus Raja).
Steroids 9:457-477.

Idler, DR, and Truscott, B. 1972. Corticosteroids in ﬁsh. Pages 126-252 in DR. Idler,
editor. Steroids in nonmammalian vertebrates. Academic Press, New York.

Kalimi, M., Shafagoj, Y., Loria, R., Padgett, D., and Regelson, W. 1994. Anti-
glucocorticoid effects of dehydroepiandrosterone (DHEA). Molecular and Cellular
Biochemistry 131:99-104.

Kumar, S., and Hedges, SB. 1998. A molecular timescale for vertebrate evolution.
Nature 392:917—920.

92

Lance, V.A., and Elsey, RM. 1986. Stress-induced suppression of testosterone secretion
in male alligators. The Journal of Experimental Zoology 239:241-246.

Lovejoy, D.A., and Balrnent, R.J. 1999. Evolution and physiology of the corticotropin-
releasing factor (CRF) family of neuropeptides in vertebrates. General and
Comparative Endocrinology 115: 1-22.

Lovejoy, D.A., and J ahan, S. 2006. Phylogeny of the corticotropin-releasing factor family
of peptides in the metazoa. General and Comparative Endocrinology 146: 1-8.

Madsen, SS. 1990. The role of cortisol and growth hormone in seawater adaptation and
development of hypoosmoregulatory mechanisms in sea trout parr (Salmo trutta
trutta). General and Comparative Endocrinology 79: 1-1 1.

Majzoub, IA. 2006. Corticotropin-releasing hormone physiology. European Journal of
Endocrinology 155:S71-S76.

McCormick, SD. 1996. Effects of growth hormone and insulin-like growth factor I on
salinity tolerance and gill Na+, K+, -ATPase in atlantic salmon (Salmo salar):
Interaction with cortisol. General and Comparative Endocrinology 101:3-11.

McCormick, S.D., Dickhoff, W.W., Duston, J ., Nishioka, R.S., and Bern, HA. 1991.
Developmental differences in the responsiveness of gill Na+, K”, -ATPase to cortisol
in salmonids. General and Comparative Endocrinology 84:308-317.

Mewes, K.R., Latz, M., Golla, H., and Fischer, A. 2002. Vitellogenin from female and
estradiol-stimulated male river lampreys (Lampetraﬂuviatilis L.). Journal of
Experimental Zoology 292:52-72.

Milston, R.H., Davis, M.W., Parker, S.J., Olla, B.L., Clements, S., and Schreck, CB.
2006. Characterization of the physiological stress response in lingcod. Transactions of
the American Fisheries Society 135:1165-1174.

Mommsen, T.P., Vijayan, M.M., and Moon, T.W. 1999. Cortisol in teleosts: dynamics,
mechanisms of action, and metabolic regulation. Reviews in Fish Biology and
Fisheries 9:211-268.

Moore, FL, and Zoeller, RT. 1985. Stress-induced inhibition of reproduction: Evidence
of suppressed secretion of LH-RH in an amphibian. General and Comparative
Endocrinology 60:252-258.

Nandi, J. and Bern, HA. 1960. Corticosteroid production by interrenal tissue of teleost
ﬁshes. Endocrinology 66:295-303.

Nelson, R.J. 2000. An introduction to behavioral endocrinology. Sinauer Associates,
Sunderland, Massachusetts.

93

Norman, A.W., and Litwack, G. 1997. Hormones. Academic Press, San Diego, CA.

Nunez, S., and Trant, J .M. 1999. Regulation of interrenal gland steroidogenesis in the
Atlantic stingray (Dasyatis sabina). Journal of Experimental Zoology 284:517-525.

Pankhurst, N.W., and Dedual, M. 1994. Effects of capture and recovery on plasma levels

of cortisol, lactate gonadal steroids in a natural population of rainbow trout. Journal
ofFish Biology 45:1013-1025.

Pepels, P.P.L.M., van Helvoort, H., Wendelaar Bonga, SE, and Balm, P.H.M. 2004.
Corticotropin-releasing hormone in the teleost stress response: rapid appearance of

the peptide in plasma of tilapia (Oreochromis mossambicus). Journal of
Endocrinology 180:425-438.

Pickering, A.D., Pottinger, T.G., Carragher, J ., and Sumpter, JP. 1987. The effects of
acute and chronic stress on the levels of reproductive hormones in the plasma of

mature male brown trout, Salmo trutta L. General and Comparative Endocrinology
68:249-259.

Quinn, M.C.J., Veillette, P.A., and Young, G. 2003. Pseudobranch and gill Na+, K+, -
ATPase activity in juvenile chinook sahnon, Oncorhynchus tshawytscha:
developmental changes and effects of growth hormone, cortisol and seawater transfer.
Comparative Biochemistry and Physiology Part A 135:249-262.

Sapolsky, R.M., Romero, L.M., and Munck, A.U. 2000. How do glucocorticoids
inﬂuence stress responses? Integrating permissive, suppressive, stimulatory, and
preparative actions. Endocrine Reviews 21:55-89.

Schreck, C.B., Bradford, C.S., Fitzpatrick, MS, and Patino, R. 1989. Regulation of the
interrenal of ﬁshes: non-classical control mechanisms. Fish Physiology and
Biochemistry 7:259-265.

Seidelin, M., Madsen, S.S., Byrialsen, A., and Kristiansen, K. 1999. Effects of insulin-
like growth factor-1 and cortisol on Na+ , K+ , -ATPase expression in osmoregulatory

tissues of brown trout (Salmo trutta). General and Comparative Endocrinology
113:331-342.

Shu, D-G., Luo, H-L., Morris, S.C., Zhang, X-L., Hu, S-X., Chen, L., Han, J ., Zhu, M.,
Li, Y., and Chen, L-Z. 1999. Lower Cambrian vertebrates from south China. Nature
402:42-46.

Sower, S.A., Moriyarna, S., Kasahara, M., Takahashi, A., Nozaki, M., Uchida, K.,
Dahlstrom, J .M., and Kawauchi, H. 2006. Identiﬁcation of sea lamprey GTHﬁ-like

cDNA and its evolutionary implications. General and Comparative Endocrinology
148:22-32.

94

Sturm, A., Bury, N., Dengreville, L., Fagart, J ., Flouriot, G., Rafestin—Oblin, M.B., and
Prunet, P. 2005. ll-Deoxycorticosterone is a potent agonist of the rainbow trout
(Oncorhynchus mykiss) mineralocorticoid receptor. Endocrinology 146:47-55.

Takahashi, A., Amemiya, Y., Sarashi, M., Sower, S.A., and Kawauchi, H. 1995.
Melanotropin and corticotropin are encoded on two distinct genes in the lamprey, the
earliest evolved extant vertebrate. Biochemical and Biophysical Research
Communications 2 1 3 2490-498.

Takahashi, A., Amemiya, Y., Nozaki, M., Sower, S.A., Joss, J ., Gorbman, A., and
Kawauchi, H. 1995. Isolation and characterization of melanotropins from lamprey
pituitary glands. International Journal of Peptide & Protein Research 46:197-204.

Takahashi, A., Yasuda, A., Sower, S.A., Kawauchi, H. 2006. Posttranslational processing
of pr00piomelanocortin family molecules in sea lamprey based on mass spectrometric
and chemical analysis. General and Comparative Endocrinology 148:79-84.

Veillette, P.A., and Young, G. 2005. Tissue culture of sockeye salmon intestine:
functional response of Na+, K“, -ATPase to cortisol. American Journal of Physiology-
Regulatory, Integrative and Comparative Physiology 288:R1598-Rl605.

Wendelaar Bonga, SE. 1997. The stress response in ﬁsh. Physiological Reviews 77:591-
625.

95

SUMMARY OF DISSERTATION

At the beginning of this research it was unknown if the ancient vertebrate, the sea
lamprey, had a stress steroid hormone. However, there were some indications that it
might exist. Research in the 1990s showed that lamprey ACTH might be an active
hormone in circulation by cloning and sequencing an ACTH homologue. Recent
evidence conﬁrmed that lamprey ACTH 1-60 is produced in the pituitary. However,
evidence is lacking that it increases in circulation or that is stimulates the release of a
stress steroid. Another piece of evidence supporting the notion that lamprey might have a
stress steroid was cloning of a corticoid receptor homologue.

Recent research has shown that lamprey possess a corticoid receptor homologue.
Researcher’s using PCR techniques ampliﬁed DNA segments from sea lamprey, which
were homologues to parts of the corticoid receptor genes in higher vertebrates. However,
the receptor had not been proven to be functional or characterized in the lamprey.

In chapter 1, the ﬁrst prediction was that lampreys have a stress steroid circulating
in the plasma. In the laboratory, various chromatography and mass spectrometry
methods were used to address this hypothesis. A large a quantity of blood was extracted
to increase the abundance of steroids for identiﬁcations. Radioimmunoassays were used
to screen the plasma for cortisol or corticosterone like steroids. These two steroids were
chosen due to their prevalence as stress steroids (glucocorticoids) in almost all
vertebrates. Once immunoreactive peaks were identiﬁed, a large quantity of the steroid
was puriﬁed for deﬁnitive identiﬁcation. LH-20 chromatography and HPLC were used

in conjunction with RIA to clean up the extract matrix before mass spectrometry. These

96

steps enabled the deﬁnitive identiﬁcation of two possible glucocorticoids in the sea
lamprey.

In chapter 2, the second prediction was that one of the two deﬁnitively identiﬁed
steroids from chapter 1 have a receptor. An important criterion to classify a hormone is
that it must have a cognate receptor. Both radioligands were used for testing of tissue
cytosol. ll-deoxycortisol, but not ll-deoxycorticosterone, bound to gill cytosol
containing glucocorticoid receptor. Our study showed that the receptor in lamprey gill
tissue had similar characteristics to glucocorticoid receptors in ﬁsh. The receptor was
found by competitive assay to be highly speciﬁc for ll-deoxycortisol. However, 11-
deoxycorticosterone was the only steroid that nearly displaced 50 percent of 11-
deoxycortisol but only at 1000 nM. Finally, this study showed that ll-deoxycortisol-
receptor complex bound to DNA-cellulose, an important step in characterization of a
receptor.

In chapter 3, the last prediction was that ll-deoxycortisol will have classic
glucocorticoid stress response and show biological effects. This study demonstrated that
adult lampreys exposed to acute stress responded with a classic glucocorticoid response.
Steroid levels were elevated and remained elevated for hours, but returned to basal levels
by 24 h. The ll-deoxycortisol implants decreased circulating androgens and estrogens in
the sea lamprey, a typical glucocorticoid effect. In addition, the implants stimulated gill
Na+, K+-ATPase activity which is mainly controlled by cortisol (glucocorticoid) in
teleosts. Evidence was shown that the hypothalamus-pituitary axis controls 11-

deoxycortisol just like cortisol in higher vertebrates. Mammalian CRH injections sharply

97

increase plasma ll-deoxycortisol. In addition, pituitary extract injections also increased
plasma ll-deoxycortisol in a dose-response manner.

The results from our research conﬁrm that one of the earliest vertebrates, the sea
lamprey, contains a stress steroid hormone (glucocorticoid). These ﬁndings are an
important step in understanding the evolution of steroid biosynthesis in the vertebrate
lineges. It is clear that ll-B hydroxylase is absent in the early vertebrates, which is an
important enzyme to in the biosynthetic pathway to make cortisol and corticosterone
which are glucocorticoids in higher vertebrates. The 11 position hydroxyl is converted to
a ketone which inactivates the steroid. Thus cortisol is converted to cortisone and
corticosterone is converted to ll-dehydrocorticosterone. An important question is; how
do the ancient vertebrates control excess stress steroid? Now research can proceed to
better understand the roles of the glucorticoid in the early vertebrates. In addition, these
results may have an impact on the management activity to control or conserve lamprey.

In the Great Lakes, management agencies have been trying to control sea lamprey
for many years. On the Paciﬁc coast, ﬁsheries management agencies are concerned
about negative impacts to Paciﬁc lampreys. Both groups now have a tool to measure or

monitor the stress steroid in lampreys, an important indicator of stress.

98

 

 

 

 
 
 
 

 

 
 

 
 

 
 

 

   

     

 

 

 

 
 

 

  
  

 

 
 

 

 

 

 

 

 

 

 
 

 

 

 

   
 

 

 

              

  
  

  

 
 

 

 

 

 

 

 

      

 

 
 

 

 

 

 
 

 
 
 
 

 

’. . . . .... . .
5“ ll . . . .

. mlliioo . . .. .
mm? . . . . . .
Biillll.2 .. . . .
ul lili . . U
til i . . .

Swli M6 . . . . . .
a.ll.llH.5 . . . . . .
Mime . . . g.
W 1-2 . . . ,
Elilo . .. ,. . .
T“ . . .. ..
mlhws . ,. .. . .. _ 5
iff“. 9 . . . . .
Milli 2 . .. . .
m 1 . . . , ., .
. . . . . ,. e
. . . v . , .. . . .1 .. . S .
. . . . . _ . . : . .. ..........A..u....1...
. , . .,. ... v T .. . .u _..,.
. .. .. . l _ . , . . , .. .. .. ...