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MICHIGAN STATE UNIVERSITY
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This is to certify that the
dissertation entitled

UNUSUAL SEX STEROIDS IN LAMPREYS

presented by

MARA BETH BRYAN

has been accepted towards fulfillment
of the requirements for the

degree in Fisheries and Wildlife and
Ph.D. Ecology, Evolutionary Biology
And Behavior

 

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UNUSUAL SEX STEROIDS IN LAMPREYS
By

Mara Beth Bryan

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
And
Program in Ecology, Evolutionary Biology, and Behavior

2004

ABSTRACT
UNUSUAL SEX STEROIDS IN LAMPREYS

By

Mara Beth Bryan

The lamprey is one of the earliest evolving extant vertebrates for which the
hypothalamus-pituitary-gonadal (HPG) axis has been shown to control reproduction. The
hypothalamic hormones, particularly gonadotropin-releasing hormone (GnRH), have
been well-characterized, but the identity and function of the gonadal steroids remain
elusive. Further knowledge regarding the proximate endocrine controls over
reproduction in the lamprey is desirable because of the lamprey’s unique place in the
phylogenic tree and the economic importance of the Species. The objective of this
dissertation research was to identify possible functional sex steroids in lampreys, develop
methods with which to measure the sex steroids, and compare sex steroid concentrations

in response to stimulation with lamprey GnRH.

This dissertation research has Shown that sea lamprey testes rapidly metabolize classical
steroids into 15a-hydroxylated derivatives in vitro, producing 15a-hydroxyprogesterone
(lSa-P) and 15a-hydroxytestosterone (15a-T). It was further demonstrated, using
radioimmunoassays (RIAS) developed as part of this dissertation research, that these
steroids are produced in vivo and circulated in the plasma. Plasma concentrations of 1501-
T are < 1 ng/ml in prespermiating male sea lampreys, but rise 2-5 times in response to
injections of GnRH, and do not show differing responses to the two types of endogenous

lamprey GnRH. Plasma concentrations of lSa-P are also < 1 ng/ml in prespermiating

male sea lampreys, but rise to average concentrations of 36 ng/ml in response to
injections of GnRH. 15(1-P concentrations rise higher in response to GnRH III than to
GnRH I, are higher at 8 h than 24 h post-injection, and higher doses of either type of
GnRH elicit higher plasma levels of 15a-P. In vitro and in vivo production of 15a-T and
15a-P was investigated in male silver lampreys (Ichthyomyzon unicuspis), chestnut
lampreys (I. casteneus), American brook lampreys (Lethenteron appendix), Pacific
lampreys (Entosphenus tridentatus), and European river lampreys (Lampetrafluviatilis).
While lSa-hydroxylated steroids were produced in vitro and in vivo in all species
examined, the response in plasma levels of 15a-hydroxylated steroids to injections of

GnRH varied among species.

This research provides a first step toward understanding the proximate controls over
reproduction in lamprey species. In sea lampreys, lSd-T is the first androgen that has
been shown to respond to hypothalamic hormones. Levels of 15a-P also increase
dramatically in response to injections of GnRH to levels far higher than those measured
previously for any steroid. It appears that 15d-hydroxylation is a common pathway in the
testes of lamprey species, but differences exist in the controls over steroidogenesis among

lamprey species.

 

 

This dissertation is dedicated to my husband, Andrew, without whose support I would

have never been able to accomplish so much.

iv

ACKNOWLEDGEMENTS
I would like to thank my graduate committee including Dr. Weiming Li, Dr. Kay
Holecamp, Dr. Kim Scribner, Dr. Robert Tempelman, Dr. Gerald Klar, and Dr.

Alexander Scott for their help guidance.

Funding was provided by the Great Lakes Fisheries Commission, the Great Lakes

Protection Fund, and National Science Foundation grant #INT-O336929.

Logistical support was provided by the US. Fish and Wildlife Service Biological Stations
in Marquette, MI and Ludington, MI; by the Hammond Bay Biological Station, which is
part of the US. Geological Survey — Biological Resources Division; and by the
Department of Fisheries and Oceans Sea Lamprey Control Centre in Sault Ste. Marie,

Ontario.

Dr. Bradley Young, Dr. Sang Seon Yun, and Mike Sieflces all contributed to this
research, and technical assistance was given by David Dewar, Jessica Glenn, and Jesse

Semeyn.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. viii

LIST OF FIGURES ................................................................................. ix

CHAPTER ONE

Classical and 15a-hydroxylated steroids in lamprey species .................................... 1
Literature cited .............................................................................. 23

CHAPTER TWO

ISa-Hydroxytestosterone produced in vitro and in vivo in the sea lamprey, Petromyzon

marinus ............................................................................................... 30
Abstract ...................................................................................... 32
Introduction ................................................................................. 33
Methods ...................................................................................... 35
Results ....................................................................................... 43
Discussion ................................................................................... 50
Literature cited .............................................................................. 57

CHAPTER THREE

ISa—Hydroxyprogesterone in male sea lampreys, Petromyzon marinas L. ................. 61
Abstract ...................................................................................... 63
Introduction ................................................................................. 64
Methods ...................................................................................... 66
Results ....................................................................................... 74
Discussion ................................................................................... 81
Literature cited .............................................................................. 88

CHAPTER FOUR

Comparison of synthesis of 15a-hydroxylated steroids in males of four North American

lamprey species ...................................................................................... 92
Abstract ...................................................................................... 94
Introduction ................................................................................. 96
Methods ...................................................................................... 98
Results ...................................................................................... 101
Discussion ................................................................................. 109
Literature cited ............................................................................ 117

CHAPTER FIVE

In vitro and in vivo production of 15a-hydroxylated steroids in adult male European river

lampreys, Lampetrafluviatilis L. ............................................................... 120
Abstract .................................................................................... 122
Introduction ................................................................................ 1 23

vi

 

 

Method5124
Results ...................................................................................... 127
Discussion ................................................................................. 133
Literature cited ............................................................................ 139

APPENDIX

lSa-Hydroxytestosterone induction by GnRH—I and GnRH-III in Atlantic and Great

Lakes sea lamprey (Petromyzon marinus L.) ................................................... 142
Abstract .................................................................................... 144
Introduction ................................................................................ 145
Methods .................................................................................... 147
Results ...................................................................................... 150
Discussion ................................................................................. 153
Literature cited ............................................................................ 159

 

 

vii

 

LIST OF TABLES
1. Studies using steroid immunoassays in lampreys ........................................... 19
2. Metabolic studies ................................................................................ 22

3. Summary of results investigating in vitro and in vivo production of 15a—hydroxylated

steroids .......................................................................................... 102

 

viii

 

LIST OF FIGURES

. Structures of hypothesized lamprey and classical steroids ................................. 10
. Lamprey phylogeny excerpted from Gill et al. 2003 ....................................... 13
. 3 H counts (dpm) in fractions following HPLC analysis of 20 pl of media from
incubation of testicular tissue with 3 H testosterone ...................................... 44
. 3 H counts (dpm) from TLC fractionation of 30 p1 of incubation media after
purification with HPLC ...................................................................... 45
. Ability of antibodies raised against different steroids to bind purified 15d-T produced
in vitro .......................................................................................... 45
. Ability of various common steroids and bile acids to displace radio-labeled lSa-T
(produced in vitro) from the antibody raised against 15a-T ............................ 46
. Assay for lSd-T (150-hydroxytestosterone) of TLC fractions from 2 pg of 15 B-T
(15B-hydroxytestosterone) standard and 200 ng of 15d-T standard .................... 47
. Expected amounts of 15a-T plotted against observed amounts in the recovery

experiment ..................................................................................... 48

9. Differences in 150t-T concentrations among sexes and stages of maturity .............. 49

10. Amount of lSa-T found in HPLC fractions from 5 ml pooled male sea lamprey

plasma based on RIA of 20 pL of each fraction .......................................... 49
l 1. Products from in vitro metabolism of 3H-P by sea lamprey testes ....................... 75
12. 3 H counts (dpm) from TLC fractionation of incubation media afier purification with
HPLC .......................................................................................... 76
13. Ability of antibodies raised against different steroids to bind purified 3H-150t-P

produced in vitro .............................................................................. 77

ix

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

Immunoreactivity to lSa-hydroxyprogesterone (lSu-P) in HPLC fractions of 1 ml
pooled plasma extract from control and GnRH-treated males .......................... 78
Responses of lSq-P plasma concentrations of prespermiating male lampreys to
exogenous GnRH ............................................................................. 79
Phylogeny of lamprey species examined in Chapter 4 .................................... 97
In vitro products of metabolism of tritiated progesterone (P) by testicular tissue of
Silver lampreys, chestnut lampreys, and American brook lampreys ................. 103
In vitro products of metabolism of tritiated testosterone (T) by testicular tissue of
Silver lampreys, chestnut lampreys, and American brook lampreys ................. 104
Immunoreactivity (ir) detected in HPLC fractions of extracted pooled plasma. . ...106
Results of GnRH-injection experiment using male Pacific lampreys ................. 108
Results investigating in vitro and in vivo production of 15d-hydroxylated steroids in
male river lampreys ......................................................................... 129
Changes in plasma steroid levels between the time of upstream migration and
spawning in river lampreys ................................................................ 130
Mean GSI of tank-held male river lampreys in different months ...................... 130
15a-P plasma concentrations of male river lampreys given two serial injections, 24 h
apart, ofGnRH (100 pg/kg) ............................................................... 131
Immunoreactivity to antibodies raised against 15a-hydroxyprogesterone (15a-P) and
15a—hydroxytestosterone (lSa-T) in HPLC-fractionated river lamprey plasma. . . 132
Radioimmunoassay of HPLC-fractionated plasma from GnRH-injected male sea

lampreys ...................................................................................... 1 50

27. Differences in lSa-hydroxytestosterone blood plasma concentrations for male sea

lampreys per site, GnRH form, dosage, and time interval ............................. 152

 

xi

 

CHAPTER ONE

Classical and 15d-hydroxylated steroids in lamprey species

 

 

Importance of knowledge regarding lamprey steroids

Information regarding lamprey steroids is vital from both basic and applied science
perspectives. Lampreys (class Cephalaspidomorphi) are one of the earliest-evolving
extant vertebrates (Hardisty and Potter 1971), and with hagfish (class Myxini) form a
monophyletic group, superclass Agnatha (Kuraku et al. 1999). Lampreys are the earliest-
evolving animals in which research has demonstrated that the hypothalamal-pituitary-
gonadal (HPG) axis controls reproduction, as it is known to do in all higher vertebrates
(reviews: Sower 1990, 1998, 2003; Sower and Kawauchi 2001). The hypothalamic
hormones in lampreys have been well-characterized (Sower 1990, 2003; Sower and
Kawauchi 2001), with two morphs of endogenous gonadotropin-releasing hormone
(GnRH) identified (Sherwood et al. 1986; Sower et al. 1993). However, research
concerning the gonadal steroids in lampreys has yielded enigmatic, and sometimes
conflicting, results. Sex steroids are hypothesized to be important to vertebrate evolution
and biocomplexity (Baker 1997, 2003, 2004), and the sex steroid-receptor system found
in lampreys may be the best living proxy for those found in ancestral vertebrates (Neidert

et al. 2001).

Besides being an important group for physiological studies based on its phylogeny,
studies of lamprey steroids have practical applications as well. Lamprey Species are in
decline over much of their range (Renaud 1997). Sea lampreys (Petromyzon marinas)
are considered delicacies in Europe (Maitland 1980) and suffer from over-fishing and
habitat destruction (Almeida et al. 2000). In the United States, the Pacific lamprey

(Entosphenus tridentatus) is a culturally important food source for Native Americans, but

 

 

populations are dwindling due to habitat loss and prevention of migration due to
hydroelectric dams (Close et al. 2002). However, in the Great Lakes, the sea lamprey is
an invasive pest that has significantly altered the food web and is subject to an expensive
international control program (Christie and Goddard 2003). Knowledge regarding
lamprey reproductive steroids may aid both conservation and control efforts (Docker et
al. 2003). Without first grasping how reproduction is controlled under natural conditions
in lampreys, it cannot be understood how adverse conditions or control techniques affect
reproduction. It also cannot be determined how a lamprey’s reproductive physiology
responds to or copes with control techniques. In addition, there has been an increased
emphasis in using and developing alternative sea lamprey control techniques, many of
which focus on the reproductive phase, in order to reduce reliance on TFM, a chemical

lamprey larvacide (Christie and Goddard 2003).

The timing and order of the evolution of steroid hormones and their receptors is a subject
of great interest and debate (e. g. Baker 2004). Studies based on nuclear receptor
sequences have led to the hypotheses that agnathans diverged from gnathostomes prior to
the evolution of a nuclear androgen receptor, and that the nuclear estrogen receptor was
the first to evolve (Thornton 2001); studies based on classical steroids (i.e., those found
in higher vertebrates; Sower 1990) in lampreys have been used to support these
hypotheses. However, if the functional steroid hormones in lampreys differ structurally
from those of other vertebrates, the results from previous studies and hypothesis based

upon them may need to be questioned.

Lamprey Life Cycle

Lamprey species can be anadromous or stay entirely in riverine habitats, and anadromous
species can give rise to landlocked forms (Hardisty and Potter 1971). In some species the
adults do not feed parasitically, and these Species are always restricted to riverine
habitats. All lamprey species are semelparous, meaning that they spawn only once before
dying. The eggs are laid in gravel nests in rivers, and hatched larvae burrow into Silt after
being carried down stream (Applegate 1950). The larvae, or ammocoetes, are filter
feeders and remain in the river bottom for 5-8 years (Potter 1980) after which the species
which undergo a parasitic stage metamorphose into adults and migrate downstream to
oceans or lakes. The lampreys feed by attaching to large fish with their sucker-like
mouths and perforating the flesh, and different species of lampreys have different
preferred attachment points depending on whether host body fluids or muscle are the
preferred food (Potter and Hillard 1987). The parasitic phase has a duration of
approximately eighteen months (Farmer 1980). Sexually mature lampreys migrate
upstream and then reproduce in streams in the spring. The upstream migration occurs in
early spring for lampreys of the genera Ichthyomyzon and Petromyzon, but occurs in
autumn for lampreys of the genera Entosphenus and Lampetra (Beamish 1980, Manion

and Hanson 1980).

Classical steroids in lampreys
Steroid levels in lampreys have often been investigated using immunoassays for classical
steroids that are commercially available (Table 1). Some of the immunoassays have been

validated by confirming the identity of the steroids chromatographically (e.g., Botticelli

1963; Kime and Larsen 1987) so that it is likely that classical steroids are produced and
circulated in lamprey plasma. Additionally, levels of classical steroids fall after
hypophysectomy (Kime and Larsen 1987; Sower and Larsen 1991), which indicates that
levels are regulated by neuroendocrine hormones, and large-dose implants of some
steroids can lead to development of secondary sex characteristics (Larsen 1974).
However, implants of classical steroids do not accelerate gonadal sexual maturation or
slow it through negative feedback loops (Larsen 1974), and exposure to exogenous

classical steroids does not affect sex determination in larvae (Docker 1992).

Progestagens. Progesterone (P) has been detected in sea lamprey plasma, and
differences in P concentrations in plasma have been detected between sexes (Linville et
al., 1987), between reproductive stages (Bolduc and Sower 1992), and in response to
GnRH (Sower et al. 1987; Sower 1989; Sower et al. 1993; Derragon and Sower 1994;
Gazourian et al. 2000). The concentrations of immunoreactive (ir)-P in most sea
lampreys are normally less than 1 ng/ml but exhibit a 2- to 5-fold increase in response to
GnRH injection (Gazourian et al. 2000). Because P can be detected by RIA in lamprey
plasma and its plasma concentrations differ in response to stimulation, it has been

suggested that P is a functional hormone in lampreys (Sower 1990).

Androgens. There is little evidence for functional classical androgens in lampreys. In
most cases, the amounts of testosterone (T) in sea lampreys were either undetectable or
very low (<1 ng/ml). Although one study did find sexual dimorphism and correlations

between maturational stage and steroid levels (Linville et al. 1987), other studies did not

support these findings (Sower et al. 1985a), and Showed that T concentrations did not
change in response to GnRH injection (Sower et al. 1985b). In other species examined, T
appears to be present, but at low concentrations that bear no relationship to stage of
maturation, gender, or treatment (Fukayama and Takahashi 1985; Kime and Larsen
1987). The only exception is the brook lamprey, Lampetra planeri Bloch, in the blood of

which Seiler et al. (1985) recorded concentrations of T of up to 15 ng/ml.

Estrogens. Estradiol (E2) has been measured using immunoassays in sea lamprey plasma
of both sexes on numerous occasions, and research regarding E2 provides the strongest
evidence of any classical steroid of it being a functional hormone (Sower 1990).
Differences in E2 concentrations in plasma have been detected between sexes (Sower et
al. 1985a; Linville et al. 1987), between reproductive stages (Sower et al. 19853; Linville
et a1. 1987; Bolduc and Sower 1992) and in response to heterologous and lamprey GnRH
(Sower 1989; Sower et al. 1983, 1985b, 1987, 1993; Derragon and Sower 1994;
Gazourian et al. 1997, 2000). Levels in GnRH-stimulated lampreys have been found to
rise as high as 12 ng/ml (Sower et al. 1983). E2 has also been measured on multiple
occasions in other lamprey species. In European river lampreys (Lampetrafluviatilis)
and Japanese river lampreys (Lampetrajaponica), plasma E2 levels have been associated
with vitellogenesis in females and also shown to increase at spawning in males
(Barranikova et al. 1995; Mewes et al. 2002; Fukayama and Takahashi 1985). The
gonadal origin of E2 is in doubt, as Kime and Larsen (1987) found that both E2 and T
plasma concentrations rose in European river lampreys after gonadectomy, and the

authors suggested that these steroids may be inactive precursors synthesized in extra-

gonadal endocrine tissues, such as the interrenal glands (and then possibly converted to

active sex hormones in the gonads).

Receptors for classicals steroids. Thus far, the only steroid in lampreys for which there
is evidence of tissue binding is E2 (Ho et a1. 1987). In this study, nuclear receptors with
an affinity to E2 were found in the nuclear and cytosolic fractions of testicular tissue in
sea lampreys. This study also found that nuclear receptors for classical androgens were
absent in the testes. Recently, it has been determined that membrane-bound steroid
receptors are found in vertebrate animals (Zhu et al. 2003a, 2003b). This mechanism of

steroid action has not been investigated in lampreys.

Sequences for partial putative nuclear receptors have been amplified using PCR-based
experiments with degenerate primers (Thornton 2001). Sequences homologous to
nuclear estrogen, progestagen, and corticoid receptors were cloned, but no androgen
receptor could be amplified. Thornton (2001) used the lack of a nuclear androgen
receptor, combined with the poor results garnered from experiments using immunoassays
for T, to hypothesize that the androgen receptor evolved through gene duplications (e.g.,
Suga et al. 1999; Escriva et al. 2002) after gnathostomes diverged from agnathans, and
therefore lampreys lack functional androgens. The binding affinities of the amplified

receptors have not yet been investigated.

Steroid metabolism in lampreys

Using radiolabeled precursors either in vitro or in vivo, classical steroids have rarely been
shown to be synthesized or metabolized to recognizable steroids as major products in
lampreys (Table 2). Weisbart and Youson (1975) incubated testicular tissue from
parasitic phase lampreys with 14C-P, and could only identify one product, which was a
very small amount of 1 l-deoxycorticosterone. In the same study, presumptive
adrenocortical tissue from larval and parasitic phase lampreys was incubated with l4C-P
and small amounts of 17-hydroxyprogesterone, androstenedione, and ll-deoxycortisol
were formed. With both the testes and presumptive adrenocortical tissues, most of the
radiolabel was converted to unknown steroids. Weisbart et al. (1977) investigated the
conversion of 3H-P in vivo in parasitic phase sea lampreys, and ll-deoxycorticosterone
was the only one of the several products that could be identified. Again, most of the
radioactivity was converted to unknown compounds. When 3H--cholesterol was used as a
substrate for in vitro incubations with adult sea lamprey testicular, ovarian, and
presumptive adrenocortical tissues, none of the steroid metabolites could be identified
(Weisbart et al. 1978). Weisbart et al. (1978) also found that testicular 3B-hydroxysteroid
dehydrogenase activity (an enzyme required for the conversion of A-5 steroids such as
pregnenolone to A-4 steroids such as P and T) was very low in comparison to other
vertebrates. Additionally, Weisbart and Idler (1970) found no evidence for the presence
of [3-4 steroids in an extract of 300 ml of plasma from prespermiating males. Callard et
al. (1980) demonstrated the presence of aromatase and Sa-reductase in the gonads by

using 3H-androstenedione as a precursor, and were able to identify small amounts of 5a-

androstenedione, estrone, and estradiol, but once again, the majority of the radioactivity

was converted to unknown products.

One explanation for the lack of identifiable products in the above studies is that lamprey
may use atypical enzymes for steroidogenic pathways. Several studies have Shown that
lamprey gonads contain an unusual lSa-steroid hydroxylase. This enzyme (or family of
enzymes) converts classical steroids and steroid precursors to more polar 15a-
hydroxylated derivatives (Fig. 1), which have so far not been identified as metabolic
products of the gonads in any other vertebrate species. The first study was by Kime and
Rafter (1981), who demonstrated that the gonads of river lamprey contain a 15-
hydroxylase that acts on P and T, although it was unclear whether T was hydroxylated in
the 15a or 150 position. Kime and Callard (1982) demonstrated that the testes of the sea
lamprey also contained enzymes that synthesized lSa-hydroxytestosterone (15a-T) and
15a-hydroxyandrostenedione from androstenedione. Kime and Larsen (1987) later
hypothesized that the lSa-hydroxylated steroids were the functional steroids in lampreys,
and that the classical steroids found in plasma were from extra-gonadal sources and not

active hormones - and were metabolized to their active form in the gonads.

More recent studies have been carried out on 15a-hydroxylated steroids in lampreys.
Lowartz et al. (2003) Showed that steroids produced in vitro and in vivo in adult lampreys
coeluted with 15a-hydroxy1ated steroids on high performance liquid chromatography
(HPLC), and later demonstrated that the same phenomenon occurs in large ammocoetes

and parasitic phase lampreys (Lowartz et al. 2004). In the course of this dissertation,

Bryan et al. (2003, 2004) confirmed the structures of lSd-T and 15a-
hydroxyprogesterone (lSa-P) as the major products of T and P in lamprey testes in vitro
using chromatographic, microchemical, and immunological techniques. lSa—T was
shown to be the only product of T, but P was converted into four different products, the
relative amounts of which varied with reproductive season. 15a-P was the only product

of P that could be identified using reference steroids.

1a. OH 1b.

 

Figure 1: Structures of hypothesized lamprey and classical steroids. Hypothesized
lamprey steroids shown are 15a-hydroxytestosterone (lSd—T, la.) and 150-
hydroxyprogesterone (15a-P4, 2a.). For comparison, the structures of the classical
hormones testosterone (1b.) and progesterone (2b.) are provided.

Plasma levels of lSa—hydroxylated steroids in sea lampreys
Although there was prior evidence for in vitro production of 15a—hydroxylated steroids,
in vivo production had not yet been investigated. The research performed for this

dissertation included developing a method to measure plasma concentrations of lSa-T

10

and 15a-P. Radioimmunoassays (RIAS) for 15a-hydroxylated steroids have been
developed and used to Show that 15a-hydroxylated steroids are produced in vivo (Table
1; Bryan et al. 2003, 2004). The radiolabel for the RIAs was produced using a large
amount of commercial tritiated T or P, and incubating it with lamprey testes so that the

endogenous lSa-hydroxylase would produce tritiated 15d-P and 15a-T.

15a-Hydroxyprogesterone. Bryan et al. (2004) used the RIA to measure the plasma
concentrations of ir-lSa-P in prespermiating males that had been given two serial
injections, 24 h apart, of either lamprey GnRH I (50, 100, or 200 pg/kg), GnRH III (50,
100, or 200 pg/kg), or saline control, with plasma being sampled 8 and 24 h after the
second injection. Plasma concentrations of ir-15a-P rose from less than 1 ng/ml to 36
ng/ml (mean of all treatments) at 8 h post-injection and declined at 24 h after injection,
but not to base levels. At 8 h after the second injection, both types of GnRH had similar
effects, but GnRH III elicited higher plasma levels at 24 h. The levels of lSa-P detected
in the treatment groups are nearly an order of magnitude greater than those measured for
any other steroid in lampreys. Levels in females were found to be less than 1 ng/ml, but
ovulating females still had significantly higher levels than preovulating females.

Sperrniating males had average plasma concentrations of 2.48 ng/ml.

ISa-Hydroxytestosterone. Bryan et al. (2003) used the RIA to measure 15a-T plasma
levels in adult lampreys and found that concentrations were all less than 1 ng/ml, but
differed significantly between sexes and between immature and mature lampreys. Young

et al. (2004a) used the RIA to measure the plasma concentrations of 15a-T in

11

prespermiating male lampreys treated with GnRH as above, and it was found that 15d-T
plasma levels rose 2-5 times in treated fish at 8 h and remained elevated at 24 h. Both
types of GnRH elicited the same response. A second study (Young et al. 2004b) used
low-dose time-release pellets of D-Arg(6)-GnRH analogs in prespermiating male
lampreys. By 6 h after the implantation of the pellets, 15a-T level were elevated 1.6-2.4
times, and remained elevated for 48 h, and after 12 h, GnRH I elicited higher responses.
Levels of 15a-T did not differ between non-treated males and males treated with bizasir
(P,P-bis(1-aziridinyl)-N-methylphosphinothioic amide). Bizasir is used as a
chemosterilant in a sterile male release program used to reduce the numbers of viable
eggs in areas which cannot be treated with lamprey larvacides (Hanson and Manion 1980,

Twohey et al. 2003).

lSa-Hydroxylated steroids in other lamprey species

Once it was determined that lSa-T and 15a-P are produced in the testes and circulated in
the plasma of sea lampreys, a study was then conducted to examine whether these
unusual steroids and steroidogenic enzymes were a unique feature of sea lampreys, or
whether they are common among lamprey species (Figure 2). The dissertation research
established that other lamprey species that evolved both before and after sea lampreys
produce lSa—P and 15a-T in vitro and in vivo (Table 1, Table 2), but how 15a-
hydroxylated steroids function within the HPG axis appears to differ among the Species
(Bryan et al. submitted, in preparation). All species examined thus far, including

members of the most ancestral genus, Ichthyomyzon, and the most derived genus,

12

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Figure 2. Lamprey phylogeny excerpted from Gill et al. 2003. The phylogeny is based
on analysis of 32 morphological, anatomical, and karyological characters to produce a
consensus tree.

Lampetra, appear to have lSa-steroid hydroxylase in adult testes (Gill et al. 2003; Kime
and Rafter 1981; Bryan et al. submitted, in preparation), and have ir—lSd-hydroxylated
steroids that coelute with standards on HPLC. However, neither 15a-P nor 15d-T plasma
concentrations change in response to either type of GnRH in male silver lampreys (I.
unicuspis), and lSa-T plasma concentrations are unaffected by either type of GnRH in
male river lampreys. Both types of GnRH cause higher plasma concentrations of lSa-P
in male river lampreys, and GnRH III, but not GnRH 1, causes higher plasma
concentrations of both 15a-P and lSa-T in pacific lampreys (Entosphenus tridentatus).

However, these increases, while statistically Significant, are still biologically small.

13

Although this difference may be explained by differences in life history, feeding ecology,
maturational stage at which experiments were conducted, and differences in experimental
protocol, these experiments are beginning to suggest that sea lamprey reproductive

physiology has diverged from that of other lamprey species.

N on-classical steroids in other aquatic vertebrates

Hagfish are the closest extant relative to lampreys, with which they form a monophyletic
group (Kuraku et al. 1999). Lamprey-like and hagfish-like species evolved in the early
Cambrian period (Shu et al. 1999) more than 500 million years ago, which supports a
molecular clock-based estimate that agnathans diverged from gnathostomes
approximately 564 million years ago (Kumar and Hedges 1998). Hagfish steroids have
not received the same attention that sea lamprey steroids have. However, Kime and
Hews (1980) and Kime et al. (1980) demonstrated that hagfish ovaries and testes possess
Sa-reductase and 613- and 7a-hydroxylases, along with other enzymes that convert P and
T into unidentified metabolites. The unusual steroids found in lampreys and hagfish may

be evolutionary artifacts.

However, many ‘non-classical’ gonadal steroids other than P, T, and E2 have been
definitively identified in teleosts (Kime 1993) though functions have only been ascribed
to a very few of them. One of the best known is 17,ZOB-dihydroxypregn-4-en-3-one
(l7,20[3-P) that is used as a maturation-inducing hormone in many teleost species and
also as a pheromone in fish of the carp family (Stacey et al. 1989). There is no evidence

that lampreys make this steroid, or any of the non-classical steroids that have been

14

studied in detail in teleost fishes (Table 2). There appears to be some variation in steroid
structure in fishes (Kime 1993) compared to other vertebrate groups, but the 15a-
hydroxylated steroids have not been found to be metabolic products of the gonads in any

other vertebrate group.

Implications, conclusions, and future work

Steroids have been linked to maturation (Nagahama 1994), courtship behavior (Stacey
and Kobayashi 1996), aggressive behavior (Elofsson et al. 2000), and migratory
movements (Munakata et al. 2001a, 2001b) in teleosts. In all vertebrates studied to date,
a common function of sex steroids is regulation of gonadal development. The gonads of
the lamprey are morphologically similar to those of teleost fish and undergo the same
developmental processes, including vitellogenesis and oocyte final maturation in females,
and spermatogenesis and sperrniation in males. In all teleost species studied thus far,
these processes are known to be under the control of sex steroids produced by the gonads:
e.g. estradiol stimulates vitellogenesis in females and spermatogonial proliferation in
males; testosterone stimulates gonadotropin production by the pituitary gland of both
sexes; ll-ketotestosterone stimulates spermatogenesis; 17,208-P stimulates oocyte final
maturation and sperrniation (review: Miura and Miura 2001). It is generally accepted that
steroids control these same processes in lampreys since the HPG axis is known to control

reproduction in lampreys.

However, further research is needed to elucidate the functions of each of the 15(1-

hydroxylated steroids. First, binding studies must be conducted to determine the

15

locations and types of receptors present for 15a-P and lSa-T. Once the target tissues and
mechanism of action of each of the 15a-hydroxylated steroids are known, it will become

easier to determine the functions of these steroids.

In teleost fish, progestagens, particularly l7a,20B-P and 1701, 200,21-
trihydroxyprogesterone, are closely linked to final maturation (N agahama 1994). In
teleosts, it has been shown not only that injection with gonadotropins or GnRH-agonists
result in spermiation and a rise in progestagens (Ueda et al. 1985; Mylonas et al. 1997;
Vermeirssen et al. 2000), but that injection with progestagens alone results in spermiation
(Ueda et al. 1985; Yueh and Chang 1997). Injections with GnRH are known to cause an
increase in lSa-P plasma concentrations (Bryan et al. 2004) and decrease the time to final
maturation (Sower 1990), but more research is needed to determine if final maturation
caused by GnRH is mediated by 15a-P. Additionally, since male lampreys release a
potent sex pheromone concurrent with the onset of spermiation (Li et al. 2002), it is
likely that the same steroidal cues are responsible for both final maturation and
pheromone release. 15a-P may be responsible for stimulating either pheromone
synthesis, the development of the secretory cells in the gills (Sieflces et al. 2003), or
pheromone release, in addition to the possibility that it stimulates final maturation and

spermiation.

Androgens have been implicated in spermatogenesis and development of secondary
sexual characteristics (Borg 1994) and aggressive behavior (Elofsson et al. 2000) in

teleosts. An additional function deserving further research in lampreys is the possible

16

involvement in migration, as androgens are known to be a factor in upstream migration in
salmon (Munataka et al. 2001a; 2001b). No classical androgens have any characteristics
of functional hormones in lampreys, and the prevailing theory is that lampreys do not use
androgens (Sower 1990; Thornton 2001). However, though its circulating levels are

relatively low, ISa-T may have androgenic functions in lampreys.

The research performed for this dissertation has furthered our knowledge of steroid
structure, functionality, and evolution in lampreys. The synthesis of 15a-P and 15a-T by
the testes has been confirmed. In vivo production and changes in circulatory levels in
response to GnRH has been demonstrated, indicating that these two steroids are likely
part of the HPG axis in sea lampreys. The enzyme pathway that produces lSa-T and
lSa-P in the testes appears to be present in all other lamprey species examined, indicating
that the lSa-hydroxylase enzyme is an ancestral trait within the Petromyzontidae family.
However, the effects of neuroendocrine hormones on the steroidogenic pathways that
produce 15 a-T and 15a-P appear to vary widely among species, which demonstrates that
lamprey species have diverged in their controls over steroidogenesis and possibly in the

functionality of ISa-hydroxylated steroids.

Much more research regarding lamprey steroids is needed to understand the significance
of the 15a-hydroxylated steroids in lampreys. We do not yet know which classical or
15a-hydroxylated steroids are functional hormones, the physiological roles of any of the
steroids, or which steroids have nuclear and/or membrane receptors. It is important to

answer these research questions, as they will further our understanding of the evolution of

17

the enzymes involved in steroidogenesis (Baker 2004), the evolution of nuclear steroid
receptors (Thornton 2001), and the evolution of ligand-receptor based methods of

regulation (Baker 2003).

18

 

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Petromyzon marinas L., excrete a sex pheromone from gill epithelia. Biol.
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Sower S.A., Larsen LG. 1990. Plasma estradiol and progesterone after hypophysectomy
and substitution with pituitary in female sea lampreys (Petromyzon marinas).
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Sower SA. 1989. Effects of lamprey gonadotropin-releasing hormone and analogs on
steroidogenesis and spermiation in male sea lampreys. Fish Physiol. Biochem. 7,
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Sower SA. 1990. Neuroendocrine control of reproduction in lampreys. Fish Physiol.
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Sower S.A., Dickoff W.W., Gorbman A., Rivier J .E., Vale W.W. 1983. Ovulatory and
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27

Sower S.A., Plisetskaya E., Gorbman A. 1985a. Changes in plasma steroid ad thyroid
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Sower S.A., Plisetskaya E., Gorbman A. 1985b. Steroid and thyroid hormone profiles
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biological properties of lamprey gonadotropin-releasing hormone in vertebrates.
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Sower S.A., Chiang Y., Lovas S., Conlon J .M. 1993. Primary structure and biological
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Sower SA. 1998. Mini-Review: Brain and pituitary hormone of lampreys, recent
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29

CHAPTER TWO

Bryan M.B, Scott A.P, Cemy 1., Yun S.S, Li W. 2003. 150t-Hydroxytestosterone
produced in vitro and in vivo in the sea lamprey, Petromyzon marinus. General and
Comparative Endocrinology 132, 418-426.

30

lSa-Hydroxytestosterone produced in vitro and in vivo in the sea lamprey,
Petromyzon marin us

Mara B. Bryan‘, Alexander P. Scott+, Ivan Cemy#, Sang Seon Yun*,
Weiming Li*

‘Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI
48824, USA; The Centre for Environment, Fisheries and Aquaculture Science, Barrack
Road, Dorset DT4 8UB, UK; #Institute of Organic Chemistry and Biochemistry,
Academy of Science of the Czech Republic, Flemingovo n. 2, 166 10 Prague 6, Czech
Republic

Running Title: lSa-Hydroxytestosterone in the sea lamprey

Corresponding author:

Weiming Li

Department of Fisheries and Wildlife
13 Natural Resources Building
Michigan State University

East Lansing, MI 48824-1222

Email: liweim@msu.edu
Tel: 517-353-9837
Fax: 517-432-1699

31

Abstract

Prior research has shown that the testes of lampreys are able to synthesize 15-
hydroxylated steroid hormones in vitro. Here we show that testes of the sea lamprey
Petromyzon marinas L. are able to convert tritiated testosterone into tritiated 15a-
hydroxytestosterone (15a-T) in high yield. The identity of the tritiated 15a -T has been
confirmed by: co-elution with standard lSa-T on high performance liquid
chromatography (HPLC); co-elution on thin layer chromatography (TLC); co-elution of
acetylated tritiated and standard 15a-T on TLC; and strong binding to an antiserum
developed against 15a-T. The strong reaction between the tritiated 15a-T and the
antiserum has been used to develop a radioimmunoassay (RIA). The RIA operates over
the range of 500 pg to 2 pg per tube; and can be applied directly to plasma samples. This
assay has been used to demonstrate that 15a-T is present in blood plasma of the sea
lampreys. The concentrations of 15a-T in captive lampreys were found to be as follows
(pg/m1; mean : sem, n): parasitic stage (reproductively immature), < 20 pg/ml, n = 7;
pre-ovulatory females, 156 i 30 pg/ml, n = 8; ovulated females, 62 i 9, n = 5; pre-
spermiating males, 275 d: 19, n = 8; spermiating males, 216 i 48, n = 8. When
spermiating male plasma was fractionated on HPLC, immunoreactivity was found
exclusively in the expected elution position of 15a-T. The biological significance of this

steroid has yet to be established.

Key words: sea lamprey, Petromyzon, endocrinology, steroid, androgen

32

Introduction

The sea lamprey, Petromyzon marinas L., is a useful model species in studies of
comparative and evolutionary endocrinology. As a member of superclass Agnatha, the
lamprey is one of the earliest evolving vertebrates still alive today. It has an anadromous
life cycle, with filter-feeding ammocoetes living in stream bottoms, parasitic phase
immature lamprey feeding in large bodies of water, and reproductive adults migrating to
tributary streams to spawn (Hardisty and Potter 1971). Additionally, information
concerning basic lamprey biology may be useful in a conservation context due to the
effort spent controlling the invasive sea lamprey population in the Laurentian Great

Lakes and conserving dwindling populations in Europe (Maitland 1980).

Previous research on lampreys has established that, similar to teleosts and higher
vertebrates, the hypothalamus-pituitary-gonadal axis, which includes two forms of
GnRH, regulates reproduction (reviews: Sower 1998; Sower and Kawauchi 2001).
However, the identity of the gonadal steroids in lampreys and their role in reproduction is
still not clear, especially with regard to the existence of androgens. There have been
several studies in which measurements have been made of plasma concentrations of
testosterone in the sea lamprey (Sower et al. 1985a, 1985b; Linville et al. 1987; Katz et
al. 1982; Weisbart et a1. 1980). In most cases, the amounts of testosterone were either
undetectable or very low (<1 ng/ml) — the exception being the paper by Weisbart et al.
(1980) in which a single value of 4.2 ng/ml was reported. This was the only study that
did not use radioimmunoassay (RIA) to measure the steroid. Despite the low

concentrations, Linville et al. (1987) found significant differences in plasma testosterone

33

concentrations between male and female lampreys and an association between
testosterone concentrations and stage of maturation. However, this finding was in
contrast to that of Sower et al. (19853), which found no differences between the sexes
and no relationship to the stage of reproduction. Sower et al. (1985b) also showed that
testosterone concentrations did not change in response to GnRH injection. The story with
other species of lamprey is very similar. Testosterone (or at least a substance or
substances cross-reacting with testosterone antibodies) is present, but at low
concentrations that bear no relationship to stage of maturation, gender, or treatment
(Fukayama and Takahashi 1985; Kime and Larsen 1987; Rinchard et al. 2000). The only
exception is the brook lamprey, Lampetra planeri Bloch, in the blood of which Seiler et

al. (1985) recorded concentrations of testosterone of up to 15 ng/ml.

Attempts have also been made to demonstrate the in viva (Weisbart et al. 1977) and in
vitro biosynthesis of testosterone by the gonads of immature (Weisbart and Youson 1975)
and mature sea lamprey (Weisbart et al. 1978). In both cases, no testosterone was found.
In the latter study, it was also reported that testicular 3B—hydroxysteroid dehydrogenase
activity (that is required for the production of 4-ene steroids such as testosterone) was

relatively low in comparison to other vertebrates.

The most likely reason for the apparent absence of testosterone in lamprey comes from a
study by Kime and Rafter (1981). These authors incubated the testes of the river
lamprey, Lampetrafluviatilis L., in vitro with either radio-labeled testosterone or radio-

labeled progesterone. In both cases, the steroids were rapidly transformed into,

34

respectively, lSB-hydroxytestosterone and 15a-hydroxyprogesterone. When Kime and
Callard (1982) incubated testes of the sea lamprey with radio-labeled androstenedione
they identified both lSa-hydroxyandrostenedione and 150t-hydroxytestosterone (15a-T).
These findings prompted the authors to predict that the functional steroids in lampreys
(whether androgens, estrogens, or progestagens) differed substantially from those of most
other vertebrates in possessing a lS-hydroxyl group. Recently there has been
confirmation that steroids produced in vivo and in vitro in sea lamprey have elution times
corresponding to lS-hydroxylated steroids (Lowartz et al. 2003). The aim of the present
study was to establish chemical evidence determining whether or not 15a-T is present in

the blood plasma of reproductively mature sea lamprey.

Our first objective was to establish whether the incubation of sea lamprey testes with
tritiated testosterone would yield 150L-T. Our second objective was to develop an
immunoassay — whether by enzyme-linked immunoassay (ELISA) or RIA - in order to
determine whether lSa-T is present in sea lamprey plasma. Our third objective was to
determine whether there were any differences between the sexes and between stages of

maturity in plasma concentrations of 15a-T.

Methods

All chemicals were obtained from Sigma Aldrich (St. Louis, MO) unless otherwise noted.
Sea lamprey were collected in streams by US. Fish and Wildlife Service employees, and
transported to either Michigan State University (East Lansing, M1) for incubation

experiments or Hammond Bay Biological Station (Millersburg, M1) for plasma sampling

35

where they were held at 10° C. Blood was obtained from the caudal vein using
heparinized syringes, held at 4° C for 20 min, and centrifuged at 2500 rpm for 20 min.

The plasma was removed and stored at -80° C.

Synthesis 0f15-hydr0xylated standards

lSa-T was prepared by a fifteen-step synthesis beginning with DHEA. The 15a-hydroxy
group was introduced using a modification of the procedure described by Hosoda et al.
(1976). A lS-hydroxyandrostane derivative was obtained and, afier change of protection,
was transformed into the testosterone series using the method described by Raggio and
Watt (1976). The final product had a melting point and optical rotation that was identical
to 15a-T obtained through microbial transformation (Tamm et al. 1963). The fully
resolved 1H NMR spectrum corresponded to established data (Kirk et al. 1990); selected
couplings in Hz: J(14a,15[3) 9.1, J(15[3,l6a) 9.7, J(15[3,16B) 3.4, J(l6a,16[3) 14.4,

J(16a,17a) 8.7, J(16B,17a) 9.1. lSB-T was prepared as in Cemy et al. (1996).

In vitro biosynthesis of tritiated I5a—T

To confirm the biosynthesis of lSa-T, testicular tissue from two freshly killed non-
spermiating adult male lampreys was finely diced with a scalpel. Replicate 500 mg
portions of diced tissue from each lamprey were placed in 50 ml polypropylene tubes
containing 10 ml L15 medium (at 6° C) and 25 uCi [1,2,6,7-3H]-testosterone. The tube
was then shaken for 4 h at 17° C. At the end of this period, the medium was removed

from each tube, filtered, and passed through an activated Sep-Pak C18 extraction

36

cartridge (Waters, Milford, MA). Each cartridge was washed with 5 ml distilled water

and eluted with 5 ml methanol. The solutions were stored at ~20° C.

In order to characterize the products of the reaction, 50 pl of methanol extract was mixed
with 10 pg each of T, 15a-T, and lSB-T (dissolved in 20 pl ethanol), dried down under a
stream of nitrogen at 45° C, redissolved in 500 pl acetonitrile/water/T F A (28/72/0.01,
v/v/v) and loaded onto a reverse-phase HPLC column (Waters, Milford, MA; 5 pm
octadecylsilane; 4.6 mm x 250 mm; fitted with a guard module). Two solvents were used
to deliver a gradient to the column. Solvent A was 0.01% TF A in distilled water and
solvent B was 70% acetonitrile and 0.01% TF A in distilled water. The pattern of
development was as follows: 0 9 10 min, 28% B; 10 9 60 min, 28% 9 100% B; 60 9
80 min, 100% B. The eluate was monitored for UV absorption with a photodiode array
detector (Waters). Fractions were collected every 1 min between 20 and 60 min into
scintillation vials. After addition of 3 ml ScintSafe Econo 1 scintillation fluid (Fisher
Scientific, Pittsburgh, PA) the vials were counted on an LS 6500 Scintillation Counter

(Beckman Coulter).

Further characterization of radioactive 15a-T was carried out by thin layer
chromatography (TLC). A 1.5 ml aliquot of methanol extract from one of the incubations
was mixed with 20 pg lSa-T and testosterone, dried down, and fractionated on HPLC as
described above. Part of the fraction (30 pl containing 50,000 dpm) corresponding to the
elution position of 15a-T was placed in a glass tube containing 10 pg lSa-T. The

solvents were removed, replaced by 100 pl pyridine and 100 pl acetic anhydride, and left

37

overnight at room temperature. A further 30 pl of the same fraction was mixed with 10
pg of 15a-T and ISB-T in a separate glass tube. The solvents in both tubes were
evaporated and replaced with 40 pl ethyl acetate. These were loaded onto separate lanes
of a TLC plate, which was developed with chloroform/ethanol (50/2; v/v). The positions
of the standards were noted by placing the plate under a UV source. The lanes were then
divided into 4 mm fractions, scraped off, placed in scintillation vials, mixed with 3 ml

scintillation fluid and counted.

Further confirmation of the identity of radioactive lSa-T was carried out by testing its
ability to bind to different dilutions of antisera developed to 15a-T-3-CMO (as described
below), 1513-T-3-CMO and 15a-P-3-CMO (and to a control serum). This required a
further 1.5 ml of the methanol extract to be fractionated on HPLC without being mixed
with any standard. The antibody dilutions were made up in 100 pl assay buffer in glass
tubes (as described below) and the radioactive 15a-T was added in a further 100 pl buffer
at the rate of 5,000 dpm per tube. After overnight incubation at 4° C, 500 pl of ice-cold
charcoal solution (50 mM sodium phosphate pH 7.4, 0.1% gelatin, 1.0% dextran-coated
charcoal) was added to each tube. The tubes were kept in ice for 20 min, and then
centrifuged at 1000 g for 12 min. The supematants were poured into 8 ml scintillation

vials, mixed with 6 ml scintillation fluid and counted.

Preparation of 1 5a-T radiolabel for use in radioimmunoassay
Approximately 1 g of minced testicular tissue was added to 12 m1 L-15 medium
containing 150 pCi tritiated testosterone and 7.3 mg NAD. After incubation at 100 C for

4 h, the medium was filtered and loaded onto an extraction cartridge as described above.

38

The lSa-T label was preferentially eluted by passing through the cartridge 10 ml of 23%
acetonitrile and 0.01% TF A in distilled water (v/v/v). This was then diluted with 20 ml
distilled water and passed through a fresh extraction cartridge, washed with 5m] distilled

water and eluted with 5 ml ethanol. The extract was stored at -20° C.

Development of antiserum

In order to develop an antiserum, a mixture of 15 mg lSa-T, 17 mg carboxymethyloxime
hydrochloride, and 25 mg sodium acetate was dissolved in 1.5 m1 methanol and left
overnight at 4° C. The methanol was then dried down under a stream of nitrogen at 45° C
and redissolved in 400 pl methanol, followed by 1 ml of water which had been adjusted
to pH 2.0 with acetic acid. The product from the reaction, lSa-T-CMO, was extracted
from this solution by shaking it twice with 4 ml ethyl acetate. The ethyl acetate was
evaporated and the extract redissolved with a small amount of methanol and then

precipitated by addition of diethyl ether. The powder was dried under vacuum.

Five mg 15a-T-CMO was dissolved in 1.5 ml of dimethylforrnamide (DMF) in a 20 ml
glass scintillation vial. The vial was placed in crushed ice within a polystyrene container
that was placed on top of a magnetic stirrer. A small magnetic flea was added to the
beaker. The ice was prevented from thawing by the occasional addition of small amounts
of liquid nitrogen to the container. With constant stirring, 3.5 pl tri-butylamine and 2.5
pl isochloroformate were added to the vial and the reaction was allowed to proceed for 40
min. In the meantime, 20 mg BSA was dissolved in 1 ml distilled water and then diluted

with 1 ml DMF and 1 drop of 2 N sodium hydroxide. This mixture, after being chilled on

39

ice, was added to the vial and left to stir for a further 3 h. After this time, the mixture
(which was slightly opaque) was centrifuged for 10 min at 1000 g. The clear supernatant
was made up to 2.5 ml with distilled water and then desalted on a PD-10 column (Nash et

al., 2000), using 3.5 ml distilled water to elute the protein fraction. The eluate was

frozen and freeze-dried.

Approximately 2 mg 150t-T-CMO-BSA was dissolved in 1 ml saline and 1 ml Freund’s
Complete Adjuvant and each injected intrademially into two rabbits. Booster injections
using the same amount of powder, but suspended in F reund’s Incomplete Adjuvant, were
given at four, six, and eight weeks following the first injection. Blood (20 ml) was
obtained eight weeks after the first injection and allowed to clot before being centrifuged

at 2500 rpm for 15 min. The serum was removed and frozen in 0.5 ml aliquots at -80° C.

Radioimmunoassay (RIA) procedure

RIAs were conducted in glass culture tubes (10 mm x 75 mm, Fisher Scientific,
Pittsburgh, PA) according to Scott et al. (1980). The assay buffer consisted of 50 mM
sodium phosphate pH 7.4, 0.2% BSA, 137 mM NaCl, 0.40 mM EDTA, 0.77 mM sodium
azide. Nine standards were made up in duplicate over the range 500 to 1.95 pg/
100pl/tube. The tubes containing unknowns also had a volume of 100 pl. Binding
reagent was made up by adding radiolabel and antiserum to 20 ml of assay buffer in
amounts such that, when 100 pl was dispensed to all tubes, each tube contained 7,000
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

40

determine the total and maximum dpm counts were also included in the assay. All tubes
were incubated overnight at 4 °C, and then placed on ice and separated with charcoal as

described above.

The specificity of the antiserum was tested by making, in 100 pl buffer, six five-fold
serial dilutions of 15a-T over a range of 10,000 to 0.64 pg/tube; of lSB—T and T over a
range of 10,000 to 3.2 pg/ tube; and llketo-testosterone, androstenedione, estradiol,
cholic acid, lSa-hydroxyprogesterone, 3keto-petromyzonol sulfate, 3keto-allochoic acid,
petromyzonol sulfate, and allochoic acid over a range of 10,000 to 400 pg/tube. A further
100 pl of buffer containing 7,000 dpm radiolabel and the lSa-T antiserum at a dilution of
1:100,000 (v/v) was added to each tube. Afier overnight incubation at 4° C they were

separated with dextran-coated charcoal as described above.

Some cross-reaction was found with ISB-T (see Results). An experiment was thus
performed to determine whether this was due to: genuine cross-reaction; possible
contamination of the standard with 15a-T; or a mixture of both. Small amounts of
standard 15a-T (200 ng) and 15B-T (2 pg) were loaded on to separate lanes of a TLC
plate (type LK6DF; Whatman International). The plate was then developed with
chloroform/ethanol (50/3; v/v) and the positions of the steroids detected by placing the
plates under a UV light source. Both lanes were divided into 5 mm sections that were
scraped into glass tubes. Assay buffer (1 ml) was added to each tube, held overnight at 4
°C, vortexed, and centrifuged briefly. Replicate 100 p1 aliquots from each tube were

assayed for 15a-T.

41

Preliminary experiments with several plasma extraction procedures (involving diethyl
ether, ethyl acetate or dichloromethane) indicated that there appeared to be little or no
interference with the assay if plasma was added directly to the tubes. An experiment was
conducted to determine whether plasma proteins that bind lSa-T are present. Pooled
plasma from parasitic phase lampreys, pre-ovulating females, ovulating females, pre-
spermiating males, and spermiating males was diluted 1:2, 1:4, 1:8, and 1:16 in assay
buffer (100 pl/tube). Radiolabel was added to all tubes (100 pl containing 7,000 dpm).
The tubes were then incubated and separated as described above for the RIA. In a second
experiment, 2 ml male plasma (pooled from several males) was mixed with 20 ng lSa-T.
The plasma was then diluted four times with the same plasma to yield a range of dilutions
of 10 to 1.25 ng/ ml. Each of these dilutions was then assayed using aliquots of 25, 50

and 100 pl aliquots. The two lower aliquots were made up to 100 p1 with assay buffer.

Inter-assay variation was determined by measuring the amount of 15a-T in the same
plasma sample (c. 2 ng/ml) in six separate assays. Intra-assay variation was determined

by measuring lSa-T in the same sample six times in the same assay.

Concentrations of 1 5a—T in lamprey plasma

Plasma was collected as described above from seven parasitic lamprey, five ovulating
females and eight each of pre-sperrniating males, pre-ovulatory females and spermiating
males. Parasitic phase lampreys were held for at least one week without feeding. For

each sample, 50 pl of plasma was assayed in duplicate.

42

To confirm that cross-reacting material in plasma had the same chromatographic
properties as lSa-T, 5 m1 pooled male sea lamprey plasma was passed through an
extraction cartridge. The steroids were eluted with 7 ml methanol and then dried with a
rotary evaporator (RE 200, Yamato, Orangesburg, NJ). The residue was subjected to
HPLC separation as described above. All fractions were assayed for 15a-T using 20 pl/

fraction.

Results

Incubation of lamprey testes with tritiated testosterone resulted in two major peaks on
HPLC (Fig. 3). The first and largest of these peaks corresponded to the elution position
of ISa-T and the second to the elution position of testosterone. The conversion rates of

3H-testosterone to ISa-T in the two testes were 85.6 % and 64.5 %.

Further confirmation of the identity of the radioactive 15a-T was obtained by running
some of the HPLC fraction on TLC. The bulk of the radioactivity eluted in the same
position as standard ISa-T (Fig. 4). Very little was associated with ISB-T.
Furthermore, when the radioactive peak was mixed with standard lSa-T and acetylated,

both radioactivity and UV absorption co-migrated.

Incubation of 1 g lamprey testis with 150 pCi tritiated T yielded approximately 75 pCi

15a-T. The dilution of antiserum that was required to bind only 50% of this radiolabel

(at 7000 dpm/ 200 pl) was 1:100,000 (v/v). The radioactive lSa-T bound strongly to the

43

15a-T antiserum, slightly to the lSB—T antiserum and not at all to the 15a-P antiserum or

control serum (Fig. 5).

1 SB-T 1 5a-T

 

 

 

 

1322263034384245505453
Fraction

Figure 3: 3 H counts (dpm) in fractions following HPLC analysis of 20 pl of media from
incubation of testicular tissue with 3 H testosterone. Arrows show where 15a-T (15a-
hydroxytestosterone), ISB-T (15 B-hydroxytestosterone), and T (testosterone) standards
elute.

44

1 5a-T 1 SB-T Acet. 1 Sat-T

50000 - ,j, 4, 1

40000 d

E 30000

a H
3
5,5 20000 -

10000 -'

0' iilIlIFfl-fl'fi

o 2 4 s 81012141618202224
Fraction No.

 

 

 

 

Figure 4: 3H counts (dpm) from TLC fractionation of 30 pl of incubation media afier
purification with HPLC. Black bars represent the purified product of incubation, and
grey bars represent the acetylated product. Arrows show the elution points of lSa-T
(lSa-hydroxytestosterone) standard, acetylated 1501-T, and lSB-T (15 B-
hydroxytestosterone) standard.

 

 

 

1'0 ‘ —.— 15a-T Antibody
~ '0' - 15a-P Antlbody
U) 0 8 .- -* - 155-T Antibody
.5 ' —v— Non-immune serum
13
.E
n 0.6 -
C
.2
t 0.4 -
O
8'
h
0. 0 2 '1 \
O.
0.0 - W h‘ "

500x 2500x 12500:: 62500:: 312500x ssszsoox
Dilution of antibody

Figure 5: Ability of antibodies raised against different steroids to bind purified ISa-T
produced in vitro. lSa-T is lSa-hydroxytestosterone, lSa-P is lSa-hydroxyprogesterone,
and ISB-T is ISB-hydroxytestosterone.

45

There was negligible cross-reaction between the 15a-T antiserum and most of the tested
steroids (Fig. 6). However, there was substantial, but non-parallel, cross-reaction of the
lSB-T (36% towards the top of the standard curve and 6.4% towards the bottom). By
running the ISB-T on TLC, it was established that a large part of the cross-reaction was
due to probable contamination of the 15 B-T standard with 150-T (Fig. 7). Some cross-

reaction was still associated, however, with the elution position of ISB-T.

 

 

00-
5°. +15a'T
: —v-- T
'o 40‘ —v -11KT
:5 —I- - Ad
5 —0- CA
a .... 3kPZS
‘L —a—- 3kACA
10‘ —o.- st
—<> - ACA
o I I I I I I I I

 

1 4 20 100 500 250010000
Amount standard (pg)

Figure 6: Ability of various common steroids and bile acids to displace radio-labeled
lSa-T (produced in vitro) from the antibody raised against lSa-T. lSa-T is 150-
hydroxytestosterone, ISB-T is 15B-hydroxytestosterone, T is testosterone, llKT is
llketo-testosterone, Ad is androstenedione, E2 is estradiol, CA is cholic acid, lSa-P is
lSa-hydroxyprogesterone, 3kPZS is 3keto-petromyzonol sulfate, 3kACA is 3keto-
allochoic acid, PZS is petromyzonol sulfate, and ACA is allochoic acid.

46

 

 

 

250' 15a-T150-r
200-
a
5150-
'5
00100-
F
50" _15a-T
£21507
0 1.1-
I I I I I I I I I I I I I I
0 2 4 6 810121416182022242628

Fraction

Figure 7: Assay for ISa-T (lSa-hydroxytestosterone) of TLC fractions from 2 pg of
15 B—T ( ISB-hydroxytestosterone) standard (filled bars) and 200 ng of 150t-T standard
(open bars). Arrows indicate the elution points of 15a-T and 15 B—T. There is only a
small positive result in the fraction containing ISB-T, indicating that actual cross-
reactivity is small. However, there are positive results in other fractions in the ISB-T
lane, indicating that the standard is contaminated.

Intra-assay variation for 150t-T was c. 5% over the middle of the standard curve. Inter-

assay variation was c. 14%.

Lamprey plasma from any sex or reproductive stage did not bind significantly to tritiated
lSa-T even at a dilution of 1:2 (v/v). Plasma dilutions yielded values near expected
when assayed in 25 pl or 50 pl volumes (Fig. 8). Assay of plasma in 100 pl volumes

yielded values slightly lower than expected.

47

 

 

1000 -
1:
a
E 100- .:
3 .-
o I
‘E 3 l
3 c
O . 10“ Q . .
E i
<
1 I I I
1 10 100 1000
Amount expected (pg)

Figure 8: Expected amounts of ISa-T plotted against observed amounts in the recovery
experiment. Known amounts of 150t-T were added to plasma, which was assayed in
different quantities and dilutions.

No ISa-T (< 20 pg/ml) was detected in plasma obtained from parasitic phase lamprey.
The concentrations of lSa-T in captive lamprey were found to be as follows (pg/ml;
mean i sem): parasitic stage, < 20; pre-ovulatory females, 156 i: 30; ovulated females,
62 2t: 9; pre—spermiating males, 275 3: 19; spermiating males, 216 d: 48. Since 150t-T was
not detected in plasma obtained from parasitic phase lamprey, they were not included in
statistical tests. Significant differences existed in plasma 150L-T concentrations among
other groups (ANOVA, F125 = 6.53, P = 0.002, Fig. 9). In pairwise comparisons (with a
Bonferroni correction) ovulating females were found to have significantly lower
concentrations than pre-sperrniating males (P = 0.002) and spermiating males (P =
0.028). After HPLC fractionation of male plasma, only one fraction contained cross-

reactive material and that was in the expected elution position of ISa-T (Fig. 10).

48

500'-

400 "' o T

 

 

 

 

:2"
O

\300"l

2 T =
'7200- 0
C3

IO

F

 

 

 

 

 

 

 

 

‘°°' i f: I

 

 

 

 

 

0 I I I I I I
P POF or PSM SM

Stage
Figure 9: Differences in 150t-T concentrations among sexes and stages of maturity. P is
parasitic phase lamprey (n = 7), OF is ovulated females (11 = 5), POF is pre-ovulatory
females (n = 8), PSM is pre-sperrniating males (n = 8), and SM is spermiated males (11 =
8). Dotted lines are means, solid center lines are medians, boxes cover the 25-75% range,
whiskers show the 10% and 90% percentiles, and dots are values either below 10% or

above 90% percentile.

 

 

 

 

15a-T
250- 1i
Ezoo- ”
.2
a; i
1 u
t 50
‘
3’
v100- ”
'1'
8
‘- 50"
0 ;::::;:::C .‘;::::;::::;:::‘;:::‘;::::;::::;::Z
20 25 30 35 40 45 50 55 00 65

Fraction

Figure 10: Amount of 15a-T found in HPLC fractions from 5 m1 pooled male sea
lamprey plasma based on RIA of 20 pL of each fraction. An arrow shows where lSa-T

elutes.

49

Discussion

This study provides conclusive evidence that the testis of the sea lamprey is able to
produce 150t-T both in vitro from exogenous testosterone and in vivo. In vitro production
of ISa-T using testosterone as a precursor has so far only been demonstrated in the river
lamprey (Kime and Rafter 1981; Golla et al. 2000), though a recent study found that the
in vitro product of testosterone co-eluted with ISa-T on HPLC (Lowartz et a1. 2003).
Kime and Callard ( 1982) found ISa-T as one of the products of incubation of sea
lamprey testes with radioactive androstenedione. Kime and Rafier (1981) obtained high
yields of ISB-T from testes of the river lamprey. However, in both the present study and
that by Kime and Callard (1982) this isomer was not formed — suggesting that the sea

lamprey lacks a lSB-hydroxylase.

The lSa-hydroxylating activity in the sea lamprey testis is so strong and specific that it
has enabled us to use lamprey testicular tissue to produce tritiated 150-T suitable for use
in RIA. This proved to be beneficial as we had originally set out to develop an ELISA
for ISa-T. However, we experienced persistent problems with replication, specificity
and generation of ‘false positives’ with the prototype ELISA. These problems were

ameliorated when we developed the RIA.

The amounts of the steroid that we have found in plasma are no higher than those of

testosterone that have previously been measured in this species. However, as opposed to

the situation with testosterone (with the exception of one study — see Introduction) there

50

are very clear differences in lSa-T concentrations between the sexes and stages of
maturation. Additionally, we have HPLC evidence that what we are measuring is in fact
lSa-T and not some other cross-reacting compound(s). There are no published data
demonstrating similar validation for RIAs of other androgens in lamprey. We have also
established (unpublished studies) that 15a-T concentrations of between 2 and 4 ng/ml are

found in the plasma of individual GnRH-injected males.

One possibility for the relatively low amounts found in the present study may be due to
the fact that the fish had been held in tanks for some weeks before they were sampled. It
is known that captivity causes a substantial reduction in sex steroid concentrations in
teleost flatfish (Vermeirssen et al. 1998). However, this possibility remains to be
established in the lamprey. Another possible reason for low amounts of ISa-T is that it is
cleared very rapidly from the plasma. The fact that there is no steroid binding activity in
the plasma would certainly be a factor that favors its rapid excretion (see discussion by

Weisbart et al. 1980).

The RIA for 15a-T has the ability to measure lSa-T over a wide range, and has low
cross-reactivity with all hormones tested, with the exception of lSB-T. However, TLC
fractionation of lSB-T with subsequent quantification of the amount of 15(1—T in the
fractions via RIA revealed that the lSB-T standard used in these analyses was heavily
contaminated and that the actual detection rate of lSB-T is very low (0.87%).

Additionally, since ISB-T does not appear to be synthesized by the testes of sea lamprey,

51

cross-reactivity is not a concern. The use of plasma, rather than a plasma extract, in the

assay did not appear to be a problem.

Although we have demonstrated that ISa-T is produced by lamprey gonads and is present
in the blood, we still cannot state whether it has a function. For this, we would need to
demonstrate the presence of receptors in one or more putative target organs - and then to
demonstrate that injection of lSa-T causes a specific biochemical, physiological or
behavioral change. Larsen (1974) has already shown that treatment of river lamprey with
exogenous testosterone causes the development of male secondary characters in the river
lamprey. This does not exclude the possibility that the testosterone exerted its activity
through conversion to 15a-T by endogenous enzyme (see discussion by Kime and Larsen

1987).

An attempt has already been made to identify DNA sequences that are common to
vertebrate androgen receptors by searching sea lamprey liver mRNA with PCR primers
(Thornton 2001). The results of this study were negative, suggesting that androgen
receptors evolved after the divergence between cyclostomes and gnathostomes, and that
lamprey therefore may not utilize androgens at all and instead use estrogens as the main
steroids in both males and females. This hypothesis is supported by findings that 1713-
estradiol is present in the blood of male sea lampreys at up to 3 ng/ml (e. g. Sower et al.
1985a) and that estrogen-binding activities were detected in both the cytosolic and
nuclear extracts of sea lamprey testes (Ho et al. 1987). However, since the search for

lamprey androgen receptors was performed using PCR primers for known (gnathostome)

52

androgen receptors, the presence of unique receptors with unique DNA sequences for

‘ ISa-hydroxylated androgens’ cannot be excluded.

If 15a-T is a functional hormone, there are several possible reasons why the lamprey may
utilize it rather than testosterone. As a parasite feeding on other vertebrates, the sea
lamprey would possibly need a mechanism to distinguish endogenous from exogenous
ingested hormones. Using a unique set of hormones, such as 15-hydroxylated steroids,
would allow lampreys an easy way to distinguish between the two. It is also possible that
the 15-hydroxylated steroids are not an evolved response to parasitism, but are simply a

primitive form of steroid hormones.

Studies of the endocrine systems of other parasites would be useful in distinguishing
between the above two hypotheses. Unfortunately, there has been very little research
performed on the steroids of other parasites, although one study did find that ticks
inactivate ingested ecdysteroids by conjugating them with fatty acid chains (Connat et al.
1986). Most studies have instead focused on the effect of parasites on host steroids.
However, based on studies of this type, it seems likely that host hormones affect parasites
feeding upon them. Lawrence (1991) found that various host steroids were found to
promote growth and/or reproductive activities in a variety of invertebrate parasites. In
certain cases, such as that of the coccidian, the parasite’s endocrine system becomes
strictly correlated with the host’s in order to synchronize reproductive activities (Porchet-
Hennere and Dugimon 1992). In situations such as that of the lamprey where the parasite
and host are very similar, having a system to distinguish endogenous from exogenous

hormones would perhaps be essential in order for the lamprey to prevent its own body

53

from responding to the host’s hormones especially since the sea lamprey feeds upon
several species with differing reproductive seasons. Further research on sea lamprey
endocrinology may help illustrate how parasites in general cope with host hormones to

prevent endocrine disruption.

In contrast, examining the endocrine systems of animals related to lamprey may help to
distinguish if 15-hydroxylated steroids are an incidental event in evolution. Hagfish are
the only other group of agnathans still alive today, though hagfish are scavengers and not
parasites, so they do not need a mechanism to cope with exogenous hormones. However,
it has been demonstrated that the hagfish produce some unusual steroids, including 5, 6,
and 7-hydroxylated hormones (Kime et al. 1980; Kime and Hews 1980). Based on
hagfish endocrinology, it appears possible that unusual steroids are due to incidental or

ancestral events in evolution.

Lampreys are not the only animals that produce steroids with a 15-hydroxyl group,
although steroids of this type are rare, and the reasons for the 15-hydroxyl group are
seldom explored. Estrogens with a 15-hydroxyl group have been found in vivo in the
peripheral plasma of the laying turkey (Brown et al. 1979) and in vitro in bovine adrenal
glands (Levy et al. 1965). Bullfrog liver slices were shown to produce 150t- and 1513-
hydroxydeoxycorticosterone in vitro (Schneider 1965). Additionally, 15-hydroxylated
steroids have been isolated from human pregnancy urine (Giannopoulos and Solomon
1967, 1970; Giannopoulos et al. 1970). Hepatic microsomes from mice have been shown
to metabolize testosterone by hydroxylation, some of which occur at the 15a or 1513

positions (Ford et al. 1975). A final instance of 15-hydroxylation has a definitive reason;

54

it was determined that in rat liver microsomes cytochrome p450 hydroxylates steroids at
several different points including the 15-C, which may make the steroids more water-

soluble in order to aid in excretion (Hrycay et al. 1976; Gustafsson 1970).

Further research is needed to elucidate if lSa-T has a function and determine the reason
for the presence of this unique hormone. It is clear that knowledge of sea lamprey
hormones can provide useful information for comparative endocrinology and the

evolution of steroid hormones.

55

Acknowledgments

Financial support for this study was provided by the Great Lakes Fisheries Commission.
We thank Hammond Bay Biological Station (U SGS-BRD) and Marquette Biological
Station (USFWS) for logistical support and Michael Siefltes for obtaining the plasma

samples used in this study.

56

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Rinchard J ., Ciereszko A., Dabrowski K., Ottobre J. 2000. Effects of gossypol on sperm
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Scott A.P., Bye V.J., Baynes S.M., Springate J RC. 1980. Seasonal variations in plasma
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59

Sower S.A., Plisetskaya E., Gorbman A. 1985a. Changes in plasma steroid ad thyroid
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Sower S.A., Plisetskaya E., Gorbman A. 1985b. Steroid and thyroid hormone profiles
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60

CHAPTER THREE

Bryan M.B., Scott A.P., Young B.A., Cemy 1., Li w. 2004. lSa-Hydroxyprogesterone
in male sea lampreys, Petromyzon marinus L. Steroids 69, 273-281.

61

ISa-Hydroxyprogesterone in male sea lampreys, Petromyzon marinas L.

Mara B. Bryan: Alexander P. Scott: Ivan Cemy#, Bradley A. Young*,
and Weiming Li*

.Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI
48824, USA; +The Centre for Environment, Fisheries and Aquaculture Science, Barrack
Road, Dorset DT4 8UB, UK; #Institute of Organic Chemistry and Biochemistry,
Academy of Science of the Czech Republic, Flemingovo n. 2, 166 10 Prague 6, Czech
Republic

Corresponding author:
Weiming Li
Department of Fisheries and Wildlife
13 Natural Resources Building
Michigan State University
East Lansing, MI 48824-1222

Email: liweim@msu.edu
Tel: 517-353-9837
Fax: 517-432-1699

Running Title: ISa-Hydroxyprogesterone in sea lamprey

62

Abstract

There is growing evidence that sea lampreys, Petromyzon marinus L., produce gonadal
steroids differing from those of other vertebrates by possessing an additional hydroxyl
group at the C15 position. Here we demonstrate that sea lamprey testes produce 150-
hydroxyprogesterone (lSa-P) in vitro when incubated with tritiated progesterone, that
150-P is present in the plasma of sea lampreys, and that plasma concentrations of
immunoreactive (ir) lSa-P rise dramatically in response to injections of gonadotropin-
releasing hormone (GnRH). The identity of the tritiated lSa-P produced in vitro was
confirmed by co-elution with standard ISa-P on high performance liquid
chromatography, co-elution with standard and acetylated ISa-P on thin layer
chromatography, and specific binding to antibodies raised against standard 15(1-P. The in
vitro conversion was used to produce tritiated ISa-P label for a radioimmunoassay (RIA),
which is able to detect 150-P in amounts as low as 2 pg per tube. The RIA has been used
to measure the plasma concentrations of lSa-P in males given two serial injections, 24 h
apart, of either lamprey GnRH I or GnRH III (50, 100, or 200 pg/kg) or saline control,
with plasma being sampled 8 and 24 h after the second injection. Plasma concentrations
of ir-lSa-P rose from < 1 ng/ml to 36 ng/ml (mean of all treatments) 8 h after injection
and declined within 24 h. This is the first time an RIA has detected such high steroid
concentrations in lampreys. This finding is suggestive of a role for 15(1—P in control of

reproduction in the sea lamprey.

Keywords: sea lamprey, lS-hydroxylation, Petromyzon, GnRH, steroid, 150-

hydroxyprogesterone

63

Introduction

As a member of superclass Agnatha, the lamprey is one of the earliest evolved extant
vertebrates (Hardisty and Potter 1971). Because of the unique position of the lamprey in
the phylogenetic tree, understanding of the mechanisms and enzymes involved in
steroidogenesis in lampreys may have important implications for our understanding of
the evolution of steroids in vertebrate species. Additionally, further knowledge regarding
lamprey reproductive processes is desirable because of the economic significance of sea
lampreys both as an invasive species targeted for control in the Great Lakes and as a

commercially important species in Europe (Maitland 1980; Almeida et al. 2000).

There is strong evidence that sea lamprey (Petromyzon marinas) gonadal steroids differ
structurally from classical steroids (i.e. those found in higher vertebrates). Investigations
into androgen production in sea lampreys have revealed that testosterone is rapidly
metabolized into 150i,17a-dihydroxy-androst-4-en-3-one (ISa-hydroxytestosterone; 150-
T) in vitro (Kime and Callard 1982; Bryan et al. 2003; Lowartz et al. 2003) and that 150-
T is produced in vivo and circulates in the plasma (Bryan et al. 2003; Lowartz et al.
2003), although at relatively low concentrations (< 1 ng/ml). When prespermiating male
lampreys are injected with exogenous gonadotropin-releasing hormone (GnRH) there is a

2- to 5- fold increase in the plasma concentrations of lSa-T (Young et al. 2004).

With respect to C21 steroids, progesterone (P) has been detected in sea lamprey plasma

using immunoassays, and differences in immunoreactive P concentrations in plasma have

been detected between sexes (Linville et al. 1987), between reproductive stages (Bolduc

64

and Sower 1992) and in response to GnRH (Sower et al. 1987; Deragon and Sower 1994;
Gazourian et a1. 2000). Very similar to the plasma concentrations of ISa-T, the
concentrations of immunoreactive P in most sea lampreys are normally less than 0.5
ng/ml but exhibit a 2- to 5-fold increase in response to GnRH injection (Gazourian et al.
2000). Because P is a hormone in mammals, can be detected by RIA in lamprey plasma,
and its plasma concentrations change in response to stimulation, it has been suggested

that P is a functional hormone in lampreys (Sower 1990).

Despite being detected by immunoassay, P has not been found to be produced in any
studies using radiolabeled precursors either in vitro or in vivo. In fact, when adult sea
lamprey testicular, ovarian, and presumptive adrenocortical tissues were incubated in
vitro with 3'H-cholesterol, none of the steroid metabolites could be identified (Weisbart et
al. 1978). However, if P is added to gonads in vitro, it does appear to be readily
metabolized. When testicular tissue from parasitic phase lampreys was incubated with
l4C-P, several products were formed of which the only one that was identified was 11-
deoxycorticosterone (Weisbart and Youson 1975). In the same study, presumptive
adrenocortical tissue from larval and parasitic phase lampreys was incubated with l"C-P
and small amounts of l7-hydroxyprogesterone, androstenedione, and ll-deoxycortisol
were formed. However, most of the radiolabel was converted to unknown steroids. The
conversion of 3H-P was investigated in vivo in parasitic phase sea lampreys, and 11-
deoxycorticosterone was the only identifiable product (Weisbart and Youson 1977).

Again, much of the radioactivity was converted to unknown compounds.

65

The lack of evidence for in vitro synthesis of P and its relatively low physiological
concentrations led Kime and Rafter (1981) to hypothesize that lampreys may use a
steroid other than P as a hormone during the final stages of gonadal maturation. These
authors incubated ovaries and testes of the river lamprey (Lampetrafluviatilis) with 3H-P
and found that the major product was 1Sa-hydroxy-pregn-4-ene-3,20-dione (150t-
hydroxyprogesterone; ISa-P). Recently, it has been shown that the elution time of a
steroid produced in vitro in male sea lampreys corresponded on high performance liquid
chromatography (HPLC) to the elution time of ISa-P (Lowartz et al. 2003). However,
the authors of this latter study did not definitively identify this steroid in the incubations,

nor provide conclusive evidence for its presence in plasma.

In order to confirm that it is lSa-P that is being made by the testis of the sea lamprey and,
more importantly, to demonstrate that it is produced in vivo, we have set out to: 1)
incubate the testis of the sea lamprey with tritiated P and then tentatively identify 15(1-P
by co-migration on HPLC and on thin layer chromatography (TLC) before and after
acetylation; 2) develop a radioimmunoassay (RIA) for ISa-P that can be applied to sea
lamprey plasma, and 3) determine whether injection of GnRH increases plasma

concentrations of ISa-P in adult male lampreys.

Methods

All chemicals were obtained from Sigma Aldrich (St. Louis, MO) unless otherwise noted.
Lamprey GnRH I and GnRH III were synthesized by Bachem Peptide Company (King of

Prussia, PA). Sea lampreys for incubation experiments (and female sea lampreys) were

66

collected dining their upstream spawning migration by US. Fish and Wildlife Service
(USFWS) employees from northern Lake Huron tributaries. Sea lampreys for the GnRH-
injection experiment were collected during their upstream spawning migration, between
May 16 and May 23, 2003, by USFWS employees using traps at the mouth of the
Cheboygan River in northern Lake Huron. The length and mass of each male for the
GnRH experiment was recorded for subsequent dosage calculations, with the average
male length (d: SEM) being 490 i 3.8 mm and average male mass being 245.8 d: 5.3 g.
Lampreys were transported to either Michigan State University (East Lansing, M1) for
incubation experiments or to the Hammond Bay Biological Station (USGS-BRD,
Millersville, M1) for plasma sampling. At Michigan State University, lampreys were
held in tanks containing 160 L of continuous-flow well water at approximately 12° C. At
Hammond Bay Biological Station, lampreys were held in tanks containing 160 L of
continuous-flow water from Lake Huron for at least one week prior to sampling.
Temperature during acclimation and experimental periods at Hammond Bay was
maintained at about 16 °C (i=1 °C). All lampreys were anesthetized with 1 : 5000
M8222 before handling. Blood was obtained from the caudal vein using heparinized

syringes, held at 4° C for 20 min, and centrifuged at 1000 x g for 20 min. The plasma

was removed and stored at -80° C.

Chemical synthesis of 15a-P
The synthesis was performed according to standard methods starting with 313-
acetoxypregna-S,l4-dien-20-one (Yoshii et al. 1977). The starting compound was

protected at position 20 in form of ethylene glycol ketal (Liu et al. 1988) and transformed

67

(Suginome et al 1992) into a 30,50-cyclo-6B-methoxy derivative. This protected
derivative was subjected to hydroboration and oxidation reactions to introduce a 15(1-
hydroxy group (Hosoda et al. 1977). The product was purified, acetylated and
deprotected in acidic medium to yield 1501-acetoxy-3 B-hydroxypregn-S-en-20-one.

Oppenauer oxidation (Raggio and Watt 1977) and deacetylation yielded ISa-P.

The physico-chemical properties of the synthetic lSa-P included a melting point of 229-
231°C (methanol), [a]D +208 (0 0.8, chloroform). For C21H3OO3 (330.5) calculated: 76.33
%C, 9.15 %H; found: 76.24 %C, 9.08 %H. The Schubert method (Schubert et al. 1962)
gave a melting point of 232°C, [a]D +218 (chloroform). The Tamm method (Tamm et al.
1963) gave a melting point of 220-228°C, [(1]D +220 (c 1.183, chloroform). The 1H NMR
spectrum (500 MHz, CDC13) was in good agreement with previously published data
(Kirk et a1. 1990) at: 1.73 (H—la), 2.04 (H-IB), 2.31 (H-Za), 2.43 (H-ZB), 5.74 (H-4), 2.35
(H-6a), 2.41 (H-6B), 1.26 (H-7a), 2.18 (H-7B), 1.78 (H-8B), 1.03 (H-9a), 1.65 (H-1 10),
1.45 (H-1 10), 1.54 (H-lZa), 2.02 (H-12B), 1.20 (H-l4a), 4.11 (H-ISB), 1.56 (H-16a),
2.78 (H-16B), 2.81 (H-17a), 0.701 (H-18), 1.204 (H-l9), 2.134 (H-21). The 13C NMR
spectrum (125.7 MHz, CDC13) was: 35.74 (C-l), 33.93 (C-2), 199.31 (C-3), 123.89 (C-
4), 170.54 (C-5), 32.69 (C-6), 31.99 (C-7), 35.20 (C-8), 53.70 (C-9), 38.57 (C-10), 20.88
(C-1 1), 38.87 (C-12), 44.55 (C-l3), 62.84 (C-14), 73.39 (C-15), 35.35 (C-l6), 60.86 (C-
17), 14.61 (C-18), 17.48 (C-19), 208.20 (C-20), 31.53 (C-21); chemical shifts correspond
to computed values from the Stothers survey (Blunt and Stothers 1977; computed data for
most affected carbons: 63.1 (C-14), 74.4 (C-15), 35.2 (C-16), 61.1 (C-17)). Electron

Impact Mass Spectrometry spectrum contained a molecular ion at m/z 330 (100%).

68

In vitro biosynthesis of tritiated 15a-P

Testicular tissue from a freshly killed adult male lamprey was finely diced in L15
medium. Fresh L15 medium (10 ml) and 0.5 g tissue was added to a 50 ml
polypropylene tube containing 25 pCi [l,2,6,7-3H]-progesterone. The tube was then
shaken for 4 h at approximately 17° C. At the end of this period, the medium was
removed, filtered, and passed through an activated Sep-Pak C18 extraction cartridge
(Waters, Milford, MA). The Sep-Pak was washed with 5 ml distilled water and eluted

with 5 ml methanol. The methanol eluate was stored at -20° C.

In order to characterize the products of the reaction, 20 pl of methanol extract was mixed
with 10 pg each of P and lSa-P (dissolved in 20 pl ethanol), dried down under a stream
of nitrogen at 45° C, fractionated using HPLC, and counted for disintegrations per minute

(dpm) (Bryan et al. 2003).

Further characterization of radioactive ISa-P was carried out by thin layer
chromatography (TLC). The remaining methanol eluate from the incubation was
fractionated using HPLC as described above. From the fraction corresponding to the
elution position of lSa-P, 50,000 dpm of the fraction was removed to each of two
microcentrifuge tubes. To both tubes, 10 pg of standard ISo-P was added and the
solvents were evaporated under nitrogen. In the first tube, 100 pl pyridine and 100 pl
acetic anhydride were added and left overnight at room temperature, after which the

solvents were again evaporated under nitrogen. The contents of both tubes were loaded

69

and developed on a TLC plate (Bryan et al. 2003). The positions of the standards were
noted by placing the plate under a UV source. The lanes were then divided into 4 mm
fractions, scraped off, placed in scintillation vials, mixed with 4 m1 scintillation cocktail

and counted.

Further confirmation of the identity of radioactive ISa-P was carried out by testing its
ability to bind to different dilutions of antisera raised against haptens for ISa-P (see
below), 15 a-T (Bryan et al. 2003) and P (provided by Dr. G. Niswender, Colorado State
University, Fort Collins, CO). The procedure was the same as that described by Bryan et

al. (2003).

Preparation of 15a-P radiolabel for use in radioimmunoassay

Radiolabel was prepared on five separate occasions. For all incubations, approximately 1
g of minced testicular tissue from a single male was added to 20 ml L15 medium
containing 150 to 200 pCi [1,2,6,7-3H]-progesterone, and the mixture was incubated for 5
h at 12° C. After all incubations, the medium was filtered and loaded onto a Sep-pak
extraction cartridge as described above. The radiolabel was further purified using HPLC
as described above, and the fractions corresponding to the elution point of lSa-P were

mixed 1 : 1 (v/v) with ethanol and stored at -20° C.

Development of antiserum
In order to develop an antiserum, ISa-P was first linked to a carboxymethyloxime

(CMO) group to make 150t-P-3-CMO. To prevent the formation of 20-(O-

70

carboxymethyl)oxime, an intermediate 3-enamine derivative was first formed (David
Kime, personal communication). To a glass tube was added 20 mg of ISa-P, 1.5 ml of
methanol and 30 ml pyrrolidine. After 5 minutes, a precipitate was formed. With
continued shaking, another 30 ml of pyrrolidine was added followed by 13 mg of
carboxymethoxylamine hemihydrochloride. The mixture was heated to 55 °C for 5 min
(during which the precipitate disappeared). The mixture was taken to dryness under a
stream of nitrogen at 45 °C, redissolved in 1 ml water and acidified with 50 ml of
concentrated HCl. The conjugate was extracted with ethyl acetate, taken to dryness,
redissolved in two drops of methanol and crystallized with diethyl ether. The total yield
was 4 mg. This was dissolved in 1.5 ml dimethyl formamide (DMF) and conjugated to

BSA as described previously (Bryan et al. 2003).

Approximately 2 mg 15a-P-3-CMO-BSA was dissolved in 1 ml saline and 1 ml Freund’s
Complete Adjuvant and injected intradermally into two rabbits. Booster injections using

the same amount of powder, but suspended in Freund’s Incomplete Adjuvant, were given
at ten weeks following the first injection. Blood (20 ml) was obtained twelve weeks after
the first injection and allowed to clot before being centrifuged at 2500 rpm for 15 min.

The serum was removed and frozen in 0.5 ml aliquots at -80° C.

RIA procedure
RIAs were conducted in glass culture tubes (10 mm x 75 mm, Fisher Scientific,

Pittsburgh, PA) as described by Bryan et al. (2003). The assay buffer consisted of 50

71

mM sodium phosphate pH 7.4, 0.2% (w/v) BSA, 137 mM NaCl, 0.40 mM EDTA, 0.77

mM sodium azide.

The specificity of the antiserum was tested by making, in 100 pl buffer, six five-fold
serial dilutions of 150t-P, P, and 17-hydroxyprogesterone over a range of 10,000 to 0.64
pg/tube; and ll-ketotestosterone, androstenedione, testosterone, ISa-T, 17B-estradiol,
cholic acid, deoxycorticosterone, 11-deoxycortisol, 3-oxo-petromyzonol sulfate, 3-oxo-
allocholic acid, petromyzonol sulfate, and allocholic acid over a range of 10,000 to 400
pg/tube. An additional 100 pl of buffer containing 5,000 dpm radiolabel and the lSa-P

antiserum at a dilution of 1 : 12,500 (v/v) was added to each tube.

Inter-assay variation was determined by measuring the amount of ISa-P in the same
plasma sample (c. 2 ng/ml) in six separate assays. Intra-assay variation was determined

by measuring ISa-P in the same sample six times in the same assay.

Blood plasma concentrations of 15a-P

The reliability of the 1501-P RIA, when applied to raw (unextracted) lamprey plasma was
tested in three ways. First, to determine specificity, 5 ml of pooled plasma from both
control and GnRH-injected fish were extracted using a Sep-pak extraction cartridge and
eluted with methanol. The eluate was evaporated with a rotary evaporator (Yamato
RE200), fractionated using HPLC as described above, and 20 pl of each fraction was
then assayed for 150-P using RIA. Second, to determine whether there might be any

proteins in plasma that might bind to 15(1-P and hence interfere with the assay, dilutions

72

of pooled plasma from both control and GnRH-treated lampreys were combined with
labeled 15a-P in the absence of antibody. Third, to establish parallelism within the assay,
standard ISa-P at a range of dilutions from 156 pg/rnl to 10 ng/ml was added to plasma
from control lampreys and assayed at volumes of 25 pl, 50 pl, and 100 pl (total volume
brought to 100 p1 with assay buffer). Plasma from GnRH-treated lampreys (8 separate

pools), pre-diluted 1 : 10 (v/v) with assay buffer, was tested the same way.

The experimental design comprised seven treatments, twelve lampreys per treatment,
with two timed injections, and two timed bleedings following a previously published
procedure (Sower 1989). Each group of 12 lampreys was injected with either 0.9% saline
(control), lamprey GnRH I (Sherwood et al. 1986; 50, 100, or 200 pg/kg body weight) or
lamprey GnRH III (Sower et al. 1993; 50, 100, or 200 pg/kg body weight). Peptides
were dissolved in 0.9% saline less than 30 min prior to administration and injected
intraperitoneally. Blood samples were collected 8 h and 24 h after the second set of
injections. Blood samples of 0.5 to 1.0 ml were collected through the caudal vein using
heparinized syringes. After centrifugation of blood samples, plasma was collected and
stored at -80 °C. For RIA analysis, plasma from control lampreys was assayed using 20
pl plasma per replicate. Plasma from treatment lampreys was assayed using 20 pl of

plasma diluted 1 : 10 (v/v) with assay buffer.

Statistical analyses were performed using the SAS system for windows, version 8. lSa-P

plasma concentrations were natural log-transformed to attain normality for all statistical

analyses. A 3-way (4x3x2) ANOVA was performed on all data investigating the main

73

effects of GnRH dosage (50, 100, or 200 pg/kg or saline), GnRH type (GnRH I or GnRH
III), and time interval (8 h or 24 h) and the potential existence of interactions. If no
meaningful interactions existed for main effects, Dunnett’s multiple comparison tests
were used to identify treatments that exceeded controls, while LSD multiple comparison
tests were used to locate differences among treatment means. If interactions between
factors were significant, the significance of differences among simple effects was

calculated.

Females and males were allowed to reach maturity in tanks containing ambient water
from Lake Huron. To determine the maturational stage of lampreys, gentle pressure was
applied to the abdomen of males or females. If this action induced emissions of milt or
eggs, the animals were classified as spermiating or ovulated. Blood samples were
obtained and processed for plasma as above for 7 each of preovulatory females, ovulated
females, and spermiating males. A t-test was used to compare lSa-P plasma

concentrations between preovulatory and ovulated females.

Results

Incubation of P with sea lamprey testes yielded up to four peaks when the media were
separated using HPLC (Fig. 11). The rate of conversion from P to any of the four peaks
differed seasonally. The average rate of conversion from P to 15a-P was 51% with SEM
of 4% during the natural spawning season (April-July) and was 14.6 at 1.4% in animals
that were kept alive artificially in cold water past the natural spawning season (August-

September).

74

1 5a-P

1

 

 

50
40 l ‘4.—' 646403

30 a

 

 

 

Percent Conversion

20 -

10 WW.-
0

50 -

4o - + 0-23-02

30 -

20 -

10 WW
0

50

40 + 94102

30

20

10
0

 

25 30 35 40 45 50 55 60 65 70 75

HPLC Fraction

Figure 11: Products from in vitro metabolism of [3H]-P by sea lamprey testes. In the
legend, the labels stand for month-day-year. The relative amounts of each product appear
to change with time. The data shown from April through August 1 represent the natural
spawning season of lampreys, while the data from August 23 and September represent
males kept in cold water to artificially keep them alive. Arrows show the elution
positions of standard ISa-hydroxyprogesterone (lSa-P) and progesterone (P).

75

Confirmation of the identity of the radioactive lSa-P was obtained by running some of
the putative HPLC-purified 15(1-P on TLC. The radioactivity co-migrated with standard

ISa-P both before and after acetylation (Fig. 12).

than.
15a4’ 15a4’
250001
IIII 1501-1D
EZZIIAcet15a43
20000:
£15000-
3
J:
I"
100001

 

 

 

 

50001
0 +%—+'$'¥I lll+L+—rflfl NH?

0 2 4 6 8 10 12 14 16 18 20 22 24 26
TLC fraction

 

Figure 12: 3 H counts (dpm) from TLC fractionation of incubation media after
purification with HPLC. Black bars represent the purified product of incubation, and
grey bars represent the acetylated product. Arrows show the elution positions of 15(1-
hydroxyprogesterone (lSa-P) and progesterone (P).

The dilution of antiserum that was required to bind 50% of lSa-P radiolabel (at 5000
dpm/ 200 pl) differed between batches of radiolabel, and ranged from 1 : 12,500 to l :

33,333 (v/v). The radioactive lSa-P bound strongly to the antisera raised against

standard lSa-P, and did not display any affinity to P, ISa-T or control antisera (Fig. 13).

76

There was negligible cross-reaction between the 150t-P antiserum and most of the tested

steroids. However, there was substantial, but non-parallel, cross-reaction with P (15.5%

for 10 ng ofP and 110% for 0.8 pg ofP).

 

1.0 -
+ 15a-P Antibody
---o--- 15a-T Antibody
o 8 ‘ —v— P Antibody
a . —-v~- Non-Immune serum
.5
E
3 0.6 ‘
r:
.2
‘5 0.4 -
Q
2
a
0.2 '
0.0 KT‘:j°———y—_ — v

 

 

 

500x 2500): 1250011 62500): 31 250011 6562500):
Dilution of antibody

Figure 13: Ability of antibodies raised against different steroids to bind purified 3H-1501-

P produced in vitro. ISa-P is lSa-hydroxyprogesterone, ISa-T is 150.-
hydroxytestosterone, and P is progesterone.

Intra-assay variation of 150t-P concentrations was 6.1%. Inter-assay variation was

13.2%.

On HPLC, there were several peaks of immunoreactive (ir)-ISa-P in plasma from both
control and GnRH-treated animals (Fig. 14). The highest peak (and the one that showed
the largest difference following GnRH-treatment) corresponded to the elution position of
lSa-P. There was no evidence for plasma protein binding of tritiated ISa-P in either

control or treatment plasma as no radiolabel remained in the supernatant after addition of

77

charcoal and centrifugation. The recovery experiment performed on pooled plasma
returned a line of expected versus observed values matching the equation of y = 1.10x +
3.00 with an r2 = 0.96 for plasma from the control group and y = 0.96x + 3.88 with an r2

= 0.93 for plasma from the treatment groups.

15 1 150'.) P

uh
O
l
A

lr-15a-P (nglml)

"I

+ GnRH-treated
-~O- -- control

 

 

 

HPLC fraction

Figure 14: Immunoreactivity to lSa-hydroxyprogesterone (ISa-P) in HPLC fractions of
1 ml pooled plasma extract from control and GnRH-treated males. The elution positions
of ISa-P and progesterone (P) are indicated with arrows.

In the injection experiment, plasma concentrations of ISa-P were higher in all GnRH-
treated animals compared to control animals at both 8 and 24 h after the second injection
(Fig. 15; P < 0.0001). All factors (dosage, injection type, and sampling time) had
significant effects. However, there was a significant interaction between the type of
GnRH injected and the sampling time, so only simple effects could be investigated

further for these two factors. Average plasma concentrations (average ng/ml i SEM) of

78

ISa-P were 36.3 i 3 at 8 h and 5.8 i 0.9 at 24 h for all GnRH-treated lampreys,

compared to 0.6 i 0.08 at 8 h and 0.7 i 0.06 at 24 h for the control groups.

 

 

 

 

 

 

 

 

 

50 50
A B
a b

40 - 40
E f a b
g 30 r a 30
z 5 ~

A. _

lg 20 4 a 20 -_ , FT 1
I" ‘IQ j

10 - 10 [— i

0 . I . I . o . ‘ . ' . .

a h 24 h GnRH I GnRH m 331an
Time Interval Injectlon Type
50 .
C

‘° ‘ a a b c
g 30 - 7,
5 " 1
q. .

20 —~
:5 l _L l

10 < 41 1

0 1 . . . , .
50 100 200 ullm

GnRH Dose (pg/kg)

Figure 15: Responses of lSa-P plasma concentrations of prespermiating male lampreys
to exogenous GnRH. Graph A shows the time at which sampling was done (levels of
control animals were not included), graph B shows the type of injection the lampreys
received (levels of control animals were not included in statistical analyses, but are in the
graph for comparison), and Graph C shows the dose of the injection. Column heights
represent group means of ISa-P plasma concentrations, and error bars represent the
standard error of the mean. Lowercase letters indicate which treatment groups were
significantly different from one another (P < 0.05) where main effects were tested for
dosage and simple effects were tested for GnRH type and time interval. ‘

Dunnett’s comparisons to controls revealed that all doses of GnRH (50, 100, or 200
ug/kg) elicited plasma concentrations significantly higher (P < 0.0001) than the saline

dose. Lampreys injected with 200 pg/kg of GnRH had significantly higher plasma

79

concentrations of lSa-P than lampreys injected with 50 or 100 pg/kg of GnRH (P <
0.05). Plasma concentrations of lSa-P did not differ between lampreys injected with 50

or 100 pg/kg of GnRH (P > 0.05).

When examining the simple effects of the type of GnRH injected, differences in lSa-P
plasma concentrations were found at both sampling times. Lampreys receiving injections
of GnRH 111 showed a trend of higher plasma concentrations of lSa-P than lampreys
receiving injections of GnRH I at the 8 h sampling time (P = 0.0560). At the 24 h
sampling time, lampreys receiving injections of GnRH 111 had significantly higher plasma

concentrations of 150-P than lamprey receiving injections of GnRH I (P < 0.0001).

When examining the simple effects of the sampling time, significant differences in 15(1-P
plasma concentrations existed between the sampling times for both types of GnRH. For
either type of GnRH, plasma levels of lSa-P were significantly higher at 8 h than at 24 h

after the second injection (P < 0.0001).

Plasma concentrations (ng/ml; mean :1: SEM, n = 7) of ISa-P in captive animals were:
preovulatory females, 0.08 :t 0.03; ovulated females, 0.69 i 0.10, spermiating males, 2.48
i: 0.89. The plasma concentrations of 15a-P in ovulated females were significantly

higher than those found in preovulatory females (P < 0.0001).

80

Discussion

Our results lend support to the hypothesis (Kime and Rafter 1981) that lampreys use 150-
P as a reproductive hormone. 150-P is readily produced both in vitro and in vivo. In
response to GnRH, the concentrations of ir-lSa-P rise far above those of any other steroid
than has previously been measured in sea lampreys. The plasma concentrations of ir-
ISa-P rose very significantly within 8 h of the second GnRH injection, and decreased
significantly, but did not fall to baseline levels, within 24 h of the second injection. The
HPLC data indicated that it was mainly 15(1—P itself, and not any of the other cross-
reacting compounds, that was responsible for the large rise in ir-lSa-P concentrations in

plasma in response to GnRH injection.

First, we confirmed that sea lamprey produce 15(1-P in vitro. Metabolism of 3H-P
changed seasonally, producing progressively more of the compounds that eluted early
from the HPLC and less in the position of lSa-P. There is not enough evidence to
indicate whether this progression is a natural phenomenon. This is because the final two
incubations in the series were carried out on testes from fish that had had their spawning
season artificially extended by keeping them in cold water. The only attempt that we
have made to identify any of these other peaks has been a single test of their ability to
bind to the lSa-P antiserum. The only peak to do so (unpublished results) was the one
eluting at 38 and 39 min — suggesting, though not proving — that this metabolite of

progesterone might also be lSa-hydroxylated.

81

The RIA for lSa-P revealed several peaks of immunoreactivity on the HPLC, of which
the largest in GnRH-injected fish was ISa-P itself. One of the peaks corresponded to the
elution position of P. This is consistent with the facts that the RIA cross-reacts with P
(albeit with poor parallelism) and that P has previously been shown to be present in the
plasma of male lampreys (see Introduction). The cross-reaction of the RIA with P is
unlikely to be a problem for physiological studies on lampreys. P concentrations, even in
GnRH-injected males, are very small relative to ISa-P plasma concentrations; baseline
levels of ir-P have been measured at 0.2 ng/ml and levels rise to 2 to 2.6 ng/ml afier
GnRH administration (Deragon and Sower 1994; Gazourian et al. 2000). HPLC of
pooled plasma samples showed that ir- 1 Sa-P in the elution position of P did not differ
greatly between treatment (0.75 ng/ml) and control plasma (0.10 ng/ml) relative to the

changes in lSa-P itself.

Two of the other immunoreactive peaks that eluted at 32-34 min and 38-39 min (cf.
elution position of one of the metabolites of tritiated P) increased a small amount in
response to GnRH. There was another immunoreactive peak at 47-48 min that showed a
more marked increase in response to GnRH injection. The presence of these other
immunoreactive peaks shows that there are at least three other, yet to be unidentified,
steroids in the plasma of the male sea lamprey. Although they appear to be present in
smaller amounts than lSa-P itself, this may be because they only exhibit partial cross-
reaction with the antibody. This is a likely scenario, but can not be established until these

steroids have been identified and synthesized. It is also not possible to determine at this

82

stage whether the immunoreactive steroids are simply metabolites of ISa-P or are any

more or less likely to be ‘hormones’ (see below).

In order to measure only ISa-P in plasma, it will be necessary to either devise a steroid
separation step or to raise a new antiserum that would hopefully have better specificity.
At this stage in research regarding lamprey sex steroids, however, we would argue that
this is not immediately necessary. Despite the interference from other steroids in the
plasma, much of the increase in immunoreactivity in GnRH-injected animals was due to
ISa-P itself. Also, standard ISa-P and plasma from these animals diluted parallel in the
RIA. Since the scale of the increase in ir-lSa-P in response to GnRH was far higher than
that recorded for any other steroid in lampreys, we believe that the RIA described in the
present paper has great value for physiological experiments in lampreys without the need

for further modification.

The change in plasma concentrations of lSa-P in response to GnRH was rapid and
significant. For both types of GnRH at all doses, 150-P concentrations were at their
highest at 8 h after the second injection, and had declined significantly, but not retumed
to basal levels, at 24 h after the second injection. A similar drop in E2 (Gazourian et al.
1997, 2000) and P (Gazourian et al. 1997) concentrations occurred between 4 h and 24 h.
However, mean concentrations of P in the latter study, even at 4 h, did not exceed 0.5

ng/ml.

83

The relatively poor dose-dependency for both peptides, at both sampling times, is almost
certainly due to the fact that even the lowest doses (50 pg/kg) were sufficient to induce
near maximum ir-lSa-P responses. Doses of 100 pg/kg and 200 pg/kg for both GnRHI
and GnRH 111 did not give a differential response in E2 plasma concentration (Gazourian
et al. 1997), though in this study doses of 200 pg/kg did elicit higher lSa-P levels than
doses of 100 pg/kg. The present study indicates that GnRH III is probably more potent
than GnRH I as ir-lSa-P concentrations tended to be higher at 8 h and were significantly
higher at 24 h in the animals that had been injected with GnRH III. This is in agreement
with the observation that GnRH III is more potent than GnRH I in stimulating

spermiation in male lampreys (Deragon and Sower 1994).

In a study by Young et al. (2004), using the same injection procedure as in the present
paper, it was found that, although plasma concentrations of ISa-T increased in response
to administration of exogenous GnRH (median level rose from 0.29 ng/ml to 1.19 ng/ml),

there were no differences between either the type of GnRH, the dose or sampling time.

In many (but not all) teleosts, as spawning time approaches, there is a switch from the
synthesis of C19 steroids (androgens) to C21 steroids (Scott et al. 1983; Barry et al. 1990).
One C21 steroid in particular, l7,20B—dihydroxy-pregn-4-en-3-one, has an important role
as an inducer of oocyte final maturation in females (Nagahama and Adachi 1985) and
sperm activation in males (Miura et al. 1992; Miura and Miura 2001). If a similar switch
in steroidogenesis takes place in lampreys, it could explain the differences seen in the

differential response of 150t-T and ir-lSa-P to GnRH administration. Our experiments

84

have mainly been carried out on adult prespermiating males very close to the onset of
spermiation, at which stage enzyme biosynthesis might naturally favor ISa-P (C21) over

ISa-T (C19) production.

Our results appear to be at variance with previous studies that indicate that lampreys have
a poor ability to produce A-4 steroids (of which 15a-P is one). Weisbart et al. (1978), for
example, found very low 3B-hydroxysteroid dehydrogenase (3 B-HSD) activity in the
testes of sexually mature sea lampreys. This enzyme is necessary for the conversion of
A-5 (pregnenolone-based) to A-4 (progesterone-based) steroids. Additionally, there was
no evidence for the presence of A-4 steroids in an extract of 300 ml of plasma from
prespermiating males (Weisbart and Idler 1970). However, neither of those studies were
carried out on GnRH-injected males. It is feasible that GnRH up-regulates 3B—HSD
activity so that 15 a-P can be formed. Additionally, non-stimulated prespermiating males
have concentrations of ISa-P that were almost certainly too low (<1 ng/ml) to have

displayed a typical A-4 steroid UV absorption signal in an extract of 300 ml plasma.

Further research will be needed to confirm if lSa-P or any of the cross-reacting steroids
are hormones. One possible theory for the existence of these steroids in the lamprey is
that P is the functional hormone but that it is rapidly metabolized into the forms that we
detect in the present study. In support of this theory is the fact that presumptive inactive
steroid metabolites are known to circulate in the plasma of teleosts at high concentrations
(Inbaraj et al. 1997; Tveiten et a1. 2000). Furthermore, these metabolites can be formed

in the gonads and not in the peripheral circulation (Kime 1993). However, in most cases

85

presumptive inactive metabolites in teleosts are conjugated steroids; the conjugation is
believed to facilitate the excretion and/or regulation of local concentrations of the steroids
(Kime 1993). It is possible that this is the reason for lSa-hydroxylation of steroids in the
lamprey testis. However, we have been carefiil to use the word ‘presumptive’ in referring
to the steroid metabolites of the teleosts. It is only ‘presumed’ that they are metabolites
in teleosts because no work has been done to determine whether they might also be

hormones.

The critical step in establishing whether 15(1-P is a hormone will be to demonstrate that
there is a receptor for this steroid in one or more tissues of the lamprey. A partial
sequence homologous to nuclear progesterone receptors has been identified in sea
lampreys (Thornton 2001), but the binding properties of the putative receptor have not
yet been examined. Even if ISa-P does not bind to this receptor, it might bind to an as
yet unidentified nuclear receptor or even to a membrane receptor such as those recently
found in a wide variety of vertebrates (Zhu et al. 2003a, 2003b); this mechanism of

hormone action has yet to be examined in lampreys.

In conclusion, we have demonstrated the production of ISa-P by the testis of the sea
lamprey; developed an RIA for this steroid; shown the presence of 15a-P and several
immunoreactive steroids in plasma; shown that the concentrations of ir-l Sa-P are
markedly elevated in response to GnRH injection; and provided a powerful new tool for

studying the endocrine control of reproduction in lampreys.

86

Acknowledgments

We thank Hammond Bay Biological Station (USGS-BRD) and Marquette Biological
Station (USFWS) for logistical support. We also thank Dr. Sang Seon Yun for his
assistance with HPLC and David Dewar for his work in the laboratory. The NMR data
were measured and interpreted by Dr. Milos Budesinsky, elemental analyses and optical
rotations were done at the Analytical Department (Dr. L. Holasova, Head), mass spectra
at Laboratory of Mass Spectrometry (Dr. K. Ubik, Head), at the Institute of Organic
Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic.
Financial support for this study was provided by the Great Lakes Fisheries Commission

and the Great Lake Protection Fund. Dr. Scott was in part supported by Defra, UK.

87

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91

CHAPTER FOUR

Bryan M.B., Young B.A., Close D.A., Semeyn J., Robinson T.C., Bayer J., Li W.
Comparison of synthesis of lSa-hydroxylated steroids in males of four North American
lamprey species. General and Comparative Endocrinology, submitted.

92

Comparison of synthesis of ISa-hydroxylated steroids in males of four North
American lamprey species

Mara B. Bryan°, Bradley A. Young“, David A; Close°, Jesse Semeyna, T. Craig
Robinsonb, Jennifer Bayer°, and Weiming Lia'

°Department of Fisheries and Wildlife, Michigan State University, 13 Natural Resources
Building, East Lansing, MI 48824; °United States Geological Survey- Columbia River
Research Laboratory, 5501A Cook-Underwood Road, Cook, WA 98605

Corresponding author:
Weiming Li
Department of Fisheries and Wildlife
13 Natural Resources Building
Michigan State University
East Lansing, MI 48824-1222

Email: liweim@msu.edu
Tel: 517-353-9837
Fax: 517-432-1699

93

Abstract

Recent studies have provided evidence that ISa-hydroxytestosterone (ISa-T) and 150-
hydroxyprogesterone (lSa-P) are produced in vitro and in vivo in adult male sea lampreys
(Petromyzon marinus), and that circulatory levels increase in response to injections with
gonadotropin-releasing hormone (GnRH). We examined four species from the
Petromyzontidae family including silver lampreys (Ichthyomyzon unicuspis), chestnut
lampreys (I. castaneus), American brook lampreys (Lethenteron appendix), and Pacific
lampreys (Entosphenus tridentata) to determine if these unusual steroids were unique to
sea lampreys or a common feature in lamprey species. In vitro production was examined
through incubations of testis with tritiated precursors, and 1501-T and 1501-P production
was confirmed in all species through co-elution with standards on both high performance
liquid chromatography (HPLC) and thin layer chromatography (TLC). In vivo
production was proven by demonstrating that HPLC-fractionated plasma had peaks of
immunoreactive lSo-T and 150t-P that coelute with standards through using previously
developed radioimmunoassays (RlAs) for 150-T and 15 a-P. The possible functionality
of lSa-T and lSa-P were further examined in silver and Pacific lampreys by
investigating the effect of injection of either type of lamprey GnRH on plasma
concentrations of lSa-T and ISa-P. Injections with exogenous GnRH did not affect
circulatory levels of either steroid in silver lampreys, and only GnRH III elicited higher
levels of both steroids in Pacific lampreys. The 150-hydroxylase enzyme(s) for steroids
appears to present in adult males of all species examined, but the question of whether
ISa-hydroxylated steroids are functional in these lamprey species, and the significance of

the 15-hydroxyl group, requires further research.

94

Keywords: lamprey, steroid, Petromyzontidae, lSa-hydroxylated

95

Introduction

There has been recent evidence to support the production of ISa-hydroxylated steroids in
vitro and in vivo in male sea lampreys, Petromyzon marinus (Kime and Callard 1982;
Bryan et al. 2003, 2004; Lowartz et al. 2003, 2004). Radioimmunoassays (RIAS) have
been developed for 15 a-hydroxytestosterone (1 Sa-T; Bryan et al. 2003) and 15(1-
hydroxyprogesterone ( lSa-P; Bryan et al. 2004) and have been used to show that plasma
concentrations of these steroids increase in response to both types of endogenous lamprey
gonadotropin-releasing hormone (GnRH; Young et al. 2004a, 2004b; Bryan et al. 2004),
and therefore may be functional hormones. Additionally, there is also research indicating
that the gonads of the European river lamprey, Lampetrafluviatilis, produce 15-
hydroxylated steroids in vitro using progesterone and testosterone as precursors, although
there is some question as to whether testosterone was hydroxylated at the a or [3 position

(Kime and Rafter 1981; Kime and Callard 1982).

In order to properly interpret the significance of lSa-hydroxylated steroids in an
evolutionary context, it must first be established when this unique pathway evolved: is it
a derived trait found only in certain lamprey lineages or is it common across lamprey
genera and therefore an ancestral trait among lamprey species? If ISa-hydroxylated
steroids are only found in Petromyzon and Lampetra species, these steroids may
represent a derived trait that evolved one or more times within the lamprey lineage.
However, if ISa-hydroxylated steroids are common to all lamprey species, it provides
evidence that the ancestral vertebrate steroids were structurally different than those

present in teleosts and higher vertebrates. There are major physiological differences,

96

including life cycles, feeding ecology, and morphology, among lamprey species (Potter
and Gill 2003), which evolved millions of years ago (Hardisty and Potter 1971).
Investigations into the presence of lSa-hydroxylated steroids across lamprey genera must
be put in the context of lamprey phylogeny (Fig. 16). However, physiological studies of
lampreys from genera other than Petromyzon are challenging because many lamprey
species are in decline (Renaud 1997), making it difficult to obtain high numbers of

individuals on which to perform extensive experiments.

—E: Ichthyomyzon unicuspis (silver)
Ichthyomyzon castaneus (chestnut)

Petromyzon marinus (sea)

 

 

 

 

Entosphenus tridentatus (Pacific)

—C Lethenteron appendix (American brook)
Lampetrafluviatilis (European river)

Figure 16. Phylogeny of lamprey species examined in Chapter 4. The phylogeny is
based on that described by Gill et al. 2003.

 

 

Our hypothesis is that lSa-hydroxylated steroids are produced in the testes and circulated
in the plasma of all lamprey species. We focused our research on genera found in North
America to make comparisons with Petromyzon. Our objectives were 1) to determine if
the lSa-hydroxylated derivatives of testosterone and progesterone are produced in vitro
in lamprey species other than P. marinas, 2) to determine if 150-P and ISa-T are

produced in vivo and present in the plasma of lamprey species other than P. marinus, and

97

3) to determine (when possible) if lSa-P and 15a-T levels in plasma change in response

to injection of GnRH I or GnRH III lamprey species.

Methods
All chemicals were obtained from Sigma unless otherwise noted. Taxonomic names are

as designated by Gill et al. (2003).

Animals. For in vitro experiments and blood drawing, silver (Ichthyomyzon unicuspis),
chestnut (I. castaneus), and American brook lampreys (Lethenteron appendix) were
caught in sea lamprey traps by US Fish and Wildlife Service personnel and transported to
Michigan State University (East Lansing, MI) in the spring of 2004. Lampreys were held

at 12 i 1 °C in flow-through tanks.

For GnRH-injection experiments, silver lampreys were caught in sea lamprey traps
during the upstream spawning migration in May near the mouth of the St. Joseph’s River
by US Fish and Wildlife Service personnel and transported to Hammond Bay Biological
Station (US Geological Survey; Millersburg, MI). The silver lampreys had an average
weight :1: standard error of 274.7 i 5.30 g. Pacific lampreys (Entosphenus tridentatus)
were collected by US Geological Survey personnel during upstream migration in
November from the John Day Dam fish ladder on the Columbia River in Oregon. Pacific
lampreys were transported to the Columbia River Research Laboratory and held under

ambient conditions, and had an average weight of 324.9 + 15.13 g.

98

All lampreys were anesthetized in 125000 MS222 prior to handling. Lampreys were
classified as spermiating if gentle pressure on the abdomen resulted in the release of milt,

and prespermiating if no milt was released (Sieflces et al. 2003)

In vitro incubations. This experiment used spermiating male silver lampreys (n = 2),
chestnut lampreys (n = 2), and American brook lampreys (n = 6). For silver and chestnut
lampreys 0.5 g of pooled gonadal tissue was used per incubation, but, because of the
small gonad size, only 0.2 g of pooled gonadal tissue was used for American brook
lampreys. The tissue was finely chopped in L-15 media added to a 50 ml conical tube
containing 5 pCi of either 3H-P or 3H-T in 10 ml L-15. One incubation was performed
per steroid per species. The tubes were shaken at 12 °C for 4 h. The tubes were then
centrifuged at 1000 g for 20 min, the media was loaded onto activated Sep-paks (Waters),

which were washed with 5 ml water and eluted with 5 ml methanol.

To identify the in vitro products, 2 ml of the methanol eluate was evaporated under
nitrogen and resuspended in high performance liquid chromotagraphy (HPLC) buffer and
fractionated using HPLC as by Bryan et a1. (2003). A 20-pl aliquot was removed from
each fraction and placed in a scintillation vial with 4 ml Safety-solve counting cocktail
(Research Products International, Mount Prospect, IL) to count disintegrations per minute
(dpm). The radioactivity in the fractions was compared to know elution points of 150-
hydroxylated and precursor standards. Percent conversion to a given product was
calculated by dividing the dpm found to coelute with the product, including more than

one fraction if necessary, by the total dpm found in all fractions.

99

Further characterization of radioactive products was carried out by thin layer
chromatography (TLC). Based on coelution with lSa-P and ISa-T on HPLC, fractions
were tentatively identified. From these fractions, an amount equivalent to 5,000 dpm was
removed and added to microcentrifuge tubes along with 10 pg of standard lSa-T or 150.-
P and evaporated under nitrogen. The contents of the tubes were loaded onto separate
lanes of a TLC plate and developed (Bryan et al. 2003). The positions of the standards
were noted by placing the plate under a UV source. The lanes were then divided into 5
mm fractions, scraped off, placed in scintillation vials, mixed with 4 ml scintillation

cocktail and counted.

1 5a-hydroxylated steroid immunoreactivity in plasma. This experiment used spermiating
male silver, chestnut, American brook, and Pacific lampreys (Entosphenus tridentatus).
Blood was obtained through the caudal vein for (n, total plasma volume) silver (2, 1.25
ml), chestnut (2, 1.25 ml), and Pacific lampreys (8, 1.5 ml), and through cardiac puncture
for American brook lampreys (6, 0.4 ml) using heparinized syringes. The blood was held
at 4 °C for 20 min, centrifuged for 20 min at 1000 g, and the plasma removed and pooled

for each species.

The pooled plasma was diluted 1:1 with 0.9% saline, passed through a 40 pM filter

(Millipore), loaded onto an activated Sep-pak, and eluted as above. The methanol eluate

was evaporated under a vacuum (CentriVap Concentrator, Labconco, Kansas City, MO)

100

and fractionated using HPLC as described previously (Bryan et al. 2003). Fractions 21-

70 were assayed for ISo-P and ISa-T using RIA as in Bryan et al. (2003, 2004).

GnRH experiments. For silver lampreys, the experiment was carried out in May 2003 at
the Hammond Bay Biological Station. For Pacific lampreys, the experiment was carried

out in February 2003 at the USGS-Columbia River Research Laboratory in Cook, WA.

There were three treatment groups with 16 lampreys per group. An initial blood sample
was taken to establish baseline steroid levels (for Pacific lampreys only). The lampreys
were given one injection with either GnRH I (Sherwood et al. 1986) or GnRH III (Sower
et al. 1993; both steroids synthesized by Bachem Peptide Company, King of Prussia, PA)
in a dose of 100 pg/kg or 0.9% saline for control animals. Blood was sampled 24 h after
the injection, and the lampreys were sacrificed so that sex could be determined. For
silver lampreys, an ANOVA was used to compare plasma steroid levels from the three
treatment groups (control, GnRH I, and GnRH III). For Pacific lampreys, t-tests were

used to compare plasma levels before and after injection for each treatment group.

Results

In vitro incubations. See Table 3 for conversion rates. All species synthesized ISa-P and
lSa-T from 3H-P and 3H-T, as determined by coelution on both HPLC (Fig. 17 and 18)
and TLC. However, all species examined also converted 3H-P into an unknown product
that eluted at 32 minutes on HPLC, and American brook lampreys converted 3H-T into an

additional unknown product as well. Silver and chestnut lampreys had similar rates of

101

conversion for both 3H-P and 3H-T, but American brook lampreys had lower rates of

conversion.

Male silver lampreys primarily metabolized P into ISa-P, but also produced steroids that
eluted at 32 min (12.3% conversion) and 36 min (5.5% conversion) on HPLC. The sole

product of T metabolism was lSa-T.

Table 3: Summary of results investigating in vitro and in vivo production of 150-
hydroxylated steroids.

 

_In vitro In vivo

 

-2, .—_..._...__ _._____.._ __........

_ % converted Co-elutes 1:1 Sa-OH in GnRH ID GnRH IIIE
(HPLC)"_. _ on TLC” __._ HPLC frastiorf

Silver
lSa-P 46.1 .1 21 NS NS
__ _15o_-T 58.97 __ \/ x/fl NS NS.“
Chestnut
lSa-P 47.6 .1 .1
- 159-T 69.3 I J - A.
American brook
ISa-P 30.4 \J \l
_ , 159-T 34.9 \/ .1
Pacific
lSa-P \/ NS P = 0.07
ISa-T .1 NS «/

 

The “\l” symbol indicates a positive result, “NS” indicates a non-statistically significant
result, and “---“ indicates that no experiment was performed.

AThe percent of the total radioactivity in the media after incubation that coelutes with
lSa-P or ISa-T on high performance liquid chromatography (HPLC).

8Whether the in vitro product that coelutes with standard on HPLC also coelutes with
standard on thin layer chromatography (TLC).

CWhether there is a peak of immunoreactivity in plasma extract that coelutes with
standard ISa-P or ISa-T on HPLC.

DWhether an injection of GnRH I (100 pg/kg) causes an increase in circulatory levels of
lSa-P or ISa-T at 24 h after injection.

EWhether an injection of GnRH III (100 pg/kg) causes an increase in circulatory levels of
ISa-P or ISa-T at 24 h after injection.

102

4o . Silver lampreys
15a-P P

.. 1 1

v

 

30 40 50 60 70

 

 

4o .
Chestnut lampreys
.1?
z 30 r
‘6
fl
.2
'0 .
E 20
E
o
I-
}. ml
0 4 . .
40 1 30 40 50 60 70
American brook lampreys
30 -

 

 

30 40 50 60 70
HPLC Fraction

Figure 17: In vitro products of metabolism of tritiated progesterone (P) by testicular
tissue of silver lampreys, chestnut lampreys, and American brook lampreys. The x-axis
is HPLC fraction and the y-axis is percent of total radioactivity. Arrows indicate the
elution points of ISa-hydroxyprogesterone (lSa-P) and P.

103

15°” T Silver lampreys

 

 

 

30 40 50 60 70

Chestnut lampreys

°/0 Total Radioactivity
N
o

 

 

 

10 -
I 1
' D
o «calla—Moo...“
30 40 50 00 70
‘01

American brook lampreys

 

 

HPLC Fraction

Figure 18: In vitro products of metabolism of tritiated testosterone (T) by testicular
tissue of silver lampreys, chestnut lampreys, and American brook lampreys. The x-axis
is HPLC fraction and the y-axis is percent of total radioactivity. Arrows indicate the
elution points of lSa-hydroxytestosterone (l Son-T) and T.

104

Male chestnut lampreys converted P into 150t-P, which was the major product, and a
product that eluted at 32-33 min (28.3% conversion) on HPLC. The sole product of T

metabolism was ISa-T.

Male American brook lampreys converted P to lSa-P, but there was more conversion
(34.1%) to a product that eluted at 32-33 min on HPLC. T was converted to lSa-T, and

was also converted (17.4%) to a product that eluted at 34 min on HPLC.

Mac-hydroxylated immunoreactivity in plasma. Pooled, fractionated plasma from
spermiating male silver, chestnut, American brook, and Pacific lampreys all had peaks of

immunoreactivity that co-eluted with ISa-P and ISa-T (Fig. 19).

For silver lampreys, immunoreactivity in the elution position of 150t-P was 1.98 ng/ml, or
31.6% of the total immunoreactivity. Immunoreactivity in the elution position of ISa-T

was 1.09 ng/ml, or 66.9% of the total immunoreactivity.

For male chestnut lamprey plasma, the total immunoreactivity to lSa-P found in all
HPLC fractions 6.04 ng/ml, with the largest amount of immunoreactivity coeluting with
lSa-P. Immunoreactivity in the elution position of lSa-P was 2.06 ng/ml, or 34.1% of
the total immunoreactivity. Immunoreactivity in the elution position of lSa—T was 2.57
ng/ml, or 96.3% of the total ir- l Sa-T, with the only peak of immunoreactivity

corresponding to the elution time of lSa-T.

105

50 -

+ Silver 1°°'P P
~-O- - Chestnut

‘0 ‘ + Am. brook
—v- - Pacifc

30-

% Total ir-1 5a-P

 

 

HPLC Fraction

1 l

% Total lr-1 5a-T

 

 

 

HPLC Fraction

Figure 19: Immunoreactivity (ir) detected in HPLC fractions of extracted pooled plasma.
The x-axis is HPLC fraction and the y-axis is percent of total immunoreactivity. The
upper graph shows ir-lSa-hydroxyprogesterone (lSa-P) in pooled plasma obtained from
male silver, chestnut, and American brook, and Pacific lampreys. The lower graph shows
ir-lSa-hydroxytestosterone (lSa-T) to the same fractions (Pacific not shown). Arrows
indicate the elution points of lSa-P, progesterone (P), ISa-T, and testosterone (T).

106

For male American brook lampreys, immunoreactivity in the elution position of ISa-P
was 5.6 ng/ml, or 36.9% of the total immunoreactivity. A second large peak of
immunoreactivity co-eluted with progesterone, and measured 3.82 ng/ml.
Immunoreactivity in the elution position of lSa-T was 8.52 ng/ml, or 49.1% of the total

ir-l Sa-T, which was the highest peak of immunoreactivity.

For male Pacific lampreys, immunoreactivity in the elution position of ISa-P was 0.75
ng/ml, or 21.7% of the total immunoreactivity. Immunoreactivity in the elution position

of 15a-T was 0.25 ng/ml, or 53% of the total ir-lSa-T.

GnRH experiments. For male silver lampreys, injections of neither type of GnRH caused
circulatory levels of ISa-P or 150t-T to rise significantly higher than those in control

animals injected with saline.

For male Pacific lampreys, there was no evidence that injections of GnRH I had an effect
on circulatory levels of either 150t-P or lSa-T. However, lampreys injected with GnRH
III showed a trend of higher plasma levels of ISa-P (P = 0.07) and significantly higher

plasma levels of ISa-T (P < 0.05) after injection (Fig. 20).

107

1:10h
-24h

150t-P (nglml)

 

 

15a-T (nglml)

 

 

 

 

GnRH l GnRH Ill control
Treatment Group

Figure 20: Results of GnRH-injection experiment using male Pacific lampreys. Pacific
lampreys were injected with GnRH I or GnRH III (100 pg/kg) or saline. Blood was
sampled prior to and 24 h alter injection, and was assayed for lSa-P (upper graph) and
ISa-T (lower graph), with the number of animals (n) indicated above each bar. Injection
with GnRH III resulted in a trend toward higher plasma levels of 15(1—P (P = 0.07) and
significantly higher lSa-T plasma levels (P < 0.05). Injection with GnRH I did not cause
a significant change in plasma levels of either steroid.

108

Discussion

lSa-Hydroxylated steroids are produced by all lamprey species included in this study.
Since this study included species from the oldest extant genus of lampreys,
Ichthyomyzon, it is likely that the common ancestor of the species examined in this study
also produced ISa-hydroxylated steroids. However, differences in production and
circulation of 1501-hydroxylated steroids are apparent among species, which may be
indicative of functional endocrine differences among lamprey species. It is important to
note that ISa-hydroxylation has not been found to be a major steroidogenic pathway in

the gonads of any other vertebrate.

All species examined formed lSa-P and 150t-T in vitro, and therefore all species
examined express lSa-hydroxylase enzyme(s) in the gonads when nearing or at
reproductive maturity. It has not yet been investigated whether there is one 150-
hydroxylase that acts on many steroids, or several different ISa-hydroxylases which are
specific to androgens, progestagens, or estrogens. The lower rates of conversion found
for both steroids by American brook lampreys may be a result of a smaller amount of
tissue used in the incubations. In silver and chestnut lampreys, ISa-T was the sole
product of testosterone metabolism, which is consistent with observations made of male
sea lampreys (Bryan et al. 2003). In American brook lampreys, an additional

unidentified product was formed, but ISa-T was still the major product.

Progesterone metabolism resulted in multiple products, some of which co-elute on HPLC

with unidentified products observed in sea lamprey (Bryan et al. 2004). The relative

109

amounts of each metabolite varied greatly among species. In sea lampreys, it was found
that the relative amounts of products changed as the reproductive season progressed, and
the differences in steroid synthesis found among species using progesterone as a

precursor may be reflective of this phenomenon. However, since these peaks are seen in

several species, it is likely that there are more unusual progestagens in lampreys that have

yet to be identified.
a“
In addition to the species examined in this study, the in vitro conversion of progesterone
and testosterone to their 15-hydroxylated derivatives has previously been demonstrated in 11
to?

 

immature adult male European river lampreys (Kime and Rafter 1981). In this study, the
major product of testosterone metabolism was reported as 15 B-T, but was later identified
as possibly being 150t-T (Kime and Callard 1982). In vivo production of 150-
hydroxylated steroids has not been investigated in this species, which is a member of the
latest genus of lamprey to evolve. Combined with the data presented here from
Ichthyomyzon species, it lends greater support to the hypothesis that ISG-hydroxylase

enzymes are pervasive in the testes of all species in the Petromyzontidae family.

All species examined in this study also had putative lSa-hydroxylated steroids produced
in vivo and circulated in the plasma, as determined by HPLC elution time and
immunoreactivity. The only major peak of ir- l Sa-T co-eluted with standard lSa-T for all
species, but ir-lSa-P was found in several fractions. Of particular interest may be the

peak that elutes at 32 min on HPLC, as all species examined thus far also convert 3H-P to

110

a product that elutes at this time. It is important to note that this peak does not coelute

with 15a-T, which elutes at 30 min.

There are several possible reasons for the different responses of different species of
lamprey to injection with GnRH. First, the injection and sampling scheme used for silver
and Pacific lamprey was different than that used on sea lamprey (Young et al. 2004a;
Bryan et al. 2004). Sea lampreys were given two serial injections, 24 h apart, and blood
was sampled 6 h and 24 h after the second injection. While ISa-T circulatory levels were
elevated at both the 6 h and 24 h sampling times (Young et al. 2004a), 150t-P circulatory
levels were elevated at 6 h and declined significantly at 24 h, but were still above
baseline levels (Bryan et al. 2004). It is possible that a single injection of GnRH did not
stimulate steroidogenesis to the same extent that two serial injections do, or that levels of

ISa-P were high at 6 h and declined by the time plasma was sampled at 24 h.

There is also a difference in the reproductive state of the animals used in these
experiments. The experiments on sea lampreys used prespermiating adult males. The
experiments on silver lampreys used adult males that were close to or already spermiated.
The Pacific lamprey experiment used very immature adult males. It is very likely that

GnRH affects steroidogenesis differently at each of these reproductive states.

Finally, it is possible that there are species-specific differences in endocrine function or

response. Silver lampreys have a life cycle similar to that of sea lampreys in which the

upstream spawning migration occurs in the spring directly prior to spawning (Manion and

111

Hanson 1980), and genus Ichthyomyzon is more closely related to genus Petromyzon than
is Entosphenus (Gill et al. 2003). Pacific lampreys cease feeding and begin spawning
migration between six months and a year prior to spawning, and overwinter in a fasting
state in streams (Beamish 1980; Close 2002). This change in life cycle likely affects the
timing of physiological events related to reproduction, and may necessitate different

patterns in expression of hormone receptors or steroidogenesis.

The two endogenous forms of sea lamprey GnRH appear to be present in all three extant
families of lamprey, including the Petromyzontidae family discussed in this paper, and
the Geotriidae and Mordaciidea found in the southern hemisphere (Sower et al. 2000),
and therefore likely evolved in an ancestral vertebrate before the families diverged. The
two types of GnRH have been shown to be equipotent in inducing final maturation and
steroidogenesis in sea lampreys, although larval sea lampreys have mainly GnRH III
present in their brains (Sower et al. 2003). In sea lampreys, GnRH III appears to cause
higher plasma levels of 150t-P in male sea lampreys (Bryan et al. 2004), but both types of
GnRH cause similar increases in 150t-T circulatory levels (Young et al. 2004a). The
results of the GnRH experiments on silver and Pacific lampreys may also point to
differences in the effects and functions of these two types of GnRH. Determination of
production of ISa-hydroxylated steroids in southern hemisphere lampreys would provide
definitive evidence of the ancestry of ISa-hydroxylase, as these families likely diverged

from Petromyzontidae in pre-Tertiary times (Gill et al. 2003).

112

The results of this study are best interpreted in the context of lamprey phylogeny. As
determined by recently discovered fossils, lamprey-like and hagfish-like species evolved
in the early Cambrian period (Shu et al. 1999) more than 500 million years ago, which
supports a molecular clock-based estimate that agnathans diverged from gnathostomes
approximately 564 million years ago (Kumar and Hedges 1998). However, times of
divergence between extant lamprey species have not been calculated. It has been
suggested that some groups of paired species are in the process of undergoing sympatric r'
speciation (Salewski 2003), and have not fully diverged into separate biological species.

For non-paired species and lampreys from different genera, it is difficult to make

 

conclusions regarding differences in physiology and phylogenetic distances.

Some of the difficulties associated with making conclusions regarding differences in
physiology and phylogenetic distances are due to controversies surrounding the
phylogeny and nomenclature of species within the Petromyzontidae family (Bailey
1980). Most of the phylogenetic relationships among lamprey species have been
determined through examination of their dentition (Hubbs and Potter 1971; Potter 1980;
Potter and Hillard 1987), but a recent study using 32 morphological characteristics and
rigorous statistical tests generated a new phylogenic tree (Gill et al. 2003). Most studies
agree that Ichthyomyzon is the ancestral genus of all holarctic lampreys, and
Ichthyomyzon unicuspis (silver lamprey) is the extant ancestral species. The genus
Petromyzon, which contains only one species, P. marinus, is derived from Ichthyomyzon,
whose two genera are monophyletic in origin (Gill et al. 2003). For the genus Lampetra,

phylogenies based on dentition (Hubbs 1971; Potter 1980) divided it into three sub-

113

genera, Entosphenus, Lethenteron, and Lampetra, though a study using two
mitochondrial genes found evidence for Entosphenus to be a separate taxon, and that the
division between Lethenteron and Lampetra does not exist (Docker et al. 1999).
However, analysis of morphological characteristics has led each of these sub-genera to be
established as a separate genus (Gill et al. 2003). Of these three groups, Entosphenus is
the most primitive, Lampetra is the most derived, and Lethenteron is intermediate. L.
fluviatilis is known to produce lS-hydroxylated steroids in vitro (Kime and Rafter 1981),
and the results of the current study support the hypothesis that the 15-hydroxylase is a
common feature in lampreys, since this enzyme is found in representatives of the most

ancestral, most derived, and intermediate genera.

Together with hagfish, lampreys form a monophyletic group (Kuraku et al. 1999),
superclass Agnatha. It has been shown that hagfish gonads hydroxylate classical steroids
at the C6 and C 7 positions in vitro (Kime and Hews 1980; Kime et al. 1980), although in
vivo production has not been investigated. The combined evidence from lampreys and
hagfish make it appear likely that ancestral vertebrates possessed steroid hydroxylases
that are no longer found in the gonads of modern vertebrates. More research is needed to
discern the reasons for both the existence of hydroxylated steroids in early vertebrates,

and for their disappearance in higher vertebrates.

There have been few studies of the reproductive physiology of lamprey species in North

American other than sea lamprey, although recorded Observances of spawning behavior

are often very similar among species (Case 1970; Manion and Hanson 1980; Cochran and

114

 

II a I an." '
' -:~":z-"“

Lyons 2003). The emphasis on sea lampreys is likely due to both its abundance in
freshwater lakes in northeastern North America and because of its economic and
ecological impacts as an invasive species. Many lamprey species around the world are in
decline (Renaud 1997) due to overfishing and habitat degradation, and obtaining
sufficient numbers of a given lamprey species for physiological experiments can be
difficult. Despite their importance as a food source to Native Americans (Close et a1.
2002) and Europeans (Maitland et al. 1980; Almeida et a1. 2000) native lamprey species
have received little attention in North America. While the sea lamprey is often targeted
as species for which hypotheses regarding early vertebrate evolution can be tested (e.g.
Thornton 2001; Baker 2004), it is important to determine whether the physiological traits

being investigated are common to all lamprey species, or are specific to sea lampreys.

115

Acknowledgements
We thank Marquette Biological Station (US Fish and Wildlife Service) and Hammond
Bay Biological Station (US Geological Survey) for logistical support, and the Great

Lakes Fisheries Commission and Great Lakes Protection Fund for funding this research.

116

 

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Bailey RM. 1980. Comments on the classification and nomenclature of lampreys - an
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Baker M.E. 2004. Co-evolution of steroidogenic and steroid-inactivating enzymes and
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M. W. and Potter, 1. C. (Eds), The Biology of Lampreys, Vol. 1. Academic Press,
New York, pp.127-206.

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Hubbs C.I., Potter LC. 1971. Distribution, phylogeny, and taxonomy. In: Hardistry, M.
W. and Potter, 1. C. (Eds), The Biology of Lampreys, Vol. 1. Academic Press,
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Kime D.E., Hews EA. 1980. Steroid biosynthesis by the ovary of the hagfish, Myxine
glutinosa. Gen. Comp. Endocrinol. 42, 71-75.

Kime D.E., Hews B.A., Rafter J. 1980. Steroid biosynthesis by the testes of the hagfish
Myxine glutinosa. Gen. Comp. Endocrinol. 41, 8-13.

Kime D.E., Callard CV. 1982. Formation of 150t-hydroxylated androgens by the testis
and other tissues of the sea lamprey, Petromyzon marinas, in vitro. Gen. Comp.
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Kime D.E., Rafter J .J ., 1981. Biosynthesis of 15-hydroxylated steroids by gonads of the
river lamprey, Lampetrafluviatilis, in vitro. Gen. Comp. Endocrinol. 44, 69-76.

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Lowartz S., Petkam R., Renaud R., Beamish F .W.H, Kime D.E., Raeside J ., Leatherland
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Lowartz S.M., Renaud R.L., Beamish F.W.H., Leatherland J .F . 2004. Evidence for 150—
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lampreys, Petromyzon marinus. Comp. Biochem. Physiol., Part B 138, 119-127.

Manion P.J., Hanson L.H. 1980. Spawning requirements and fecundity of lampreys
from the upper three Great Lakes. Can. J. Fish. Aquat. Sci. 37, 1635-1640.

Potter LC. 1980. The Petromyzoniforrnes with particular reference to paired species.
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Potter I.C., Hillard R.W. 1987. A proposal for the functional and phylogenetic
significance of differences in the dentition of lampreys (Agnatha:
Petromyzoniformes). J. Zool., Lond 212, 713-737.

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Renaud J .E. 1997. Conservation status of northern hemisphere lampreys
(Petromyzontidae). J. Applied Ichthyology 13, 143-148.

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speciation in sympatry? J. Fish. Biol. 63, 267-279.

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Li Y., Chen L-Z. 1999. Lower Cambrian vertebrates from south China. Nature
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Sieflres M.J, Scott A.P., Zielinski B., Yun S.S., Li W. 2003. Male sea lampreys,
Petromyzon marinas L., excrete a sex pheromone from gill epithelia. Biol.
Reprod. 69, 125-132.

Sower SA. 2003. The endocrinology of reproduction in lampreys and applications for
male lamprey sterilization. J. Great Lakes Res. 29 (Suppl 1), 50-65.

Sower S.A., McGregor A.J., Mateme O.L.J., Chase C., Potter 1., Joss J. 2000. Evidence
for lamprey GnRH-I and GnRH-III-like molecules in the brains of the southern

hemisphere lampreys Geotria australis and Mordacia mordax. Gen. Comp.
Endocrinol. 120, 168-175.

Thornton J .W. 2001. Evolution of vertebrate steroid receptors from an ancestral
estrogen receptor by ligand exploitation and serial genome expansions. PNAS 98,
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Young B.A., Bryan M.B., Sower S.A., Scott A.P., Li W. 2004a. 150t-
hydroxytestosterone induction by GnRH-I and GnRH-III in Atlantic and Great
Lakes sea lamprey (Petromyzon marinus L.). Gen. Comp. Endocrinol. 136, 276-
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Young B.A., Sower S.A., Bryan M.B., Li W. 2004b. The effect of chemosterilization on

sex steroid production in male sea lampreys. Trans. Amer. Fish. Soc., 133,
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119

CHAPTER FIVE

Bryan M.B., Scott A.P., Lucas M.C., Li W. In vitro and in vivo production of 15(1-
hydroxylated steroids in adult male European river lampreys, Lampetrafluviatilis L.

120

 

In vitro and in vivo production of lSa-hydroxylated steroids in adult male European
river lampreys, Lampetrafluviatilis L.

M.B. Bryan‘, A.P. Scott2, M.C. Lucas°, W. Li'“

1Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI
48824, U.S.A.; 2The Centre for Environment, Fisheries and Aquaculture Science,
Barrack Road, Dorset, DT4 8UB, U.K.; 3School of Biological and Biomedical Sciences,
University of Durham, South Road, Durham, DH1 3LE, U.K.

. Corresponding author; Tel.: 517 353 9837; Fax: 517 432 1699; email: liweim@msu.edu

Running title: 15-OH Steroids in European River Lampreys

121

Abstract

The European river lamprey, Lampetrafluviatilis, is a member of the latest lamprey
genus to evolve, and therefore represents a key group for examining the evolution of
features within the Petromyzontidae family. Like the males of several North American
species of lampreys, mature river lamprey testes producelSo-hydroxytestosterone (lSa-T)
and 15a-hydroxyprogestone (lSa-P) in vitro. Males circulate immunoreactive (ir) ISa-T
and 150-P in the plasma. Plasma concentrations of ISa-P increase as the reproductive
season approaches, and levels of ISo-T do not change. Plasma concentrations of 150t-P
increase in response to injections of GnRH I or GnRH III, and plasma concentrations of
ISa-T do not change in response to either type of GnRH. This finding provides further
evidence that 15a-steroid hydroxylase in the testes is a common feature of holarctic
lampreys, but raises questions regarding differences in potential functionality of 150-

hydroxylated steroids and the controls over steroidogenesis among lamprey species.

Keywords: river lamprey; Lampetra; ISa-hydroxylated; Petromyzontidae.

122

Introduction

The European river lamprey (Lampetrafluviatilis) is considered to belong to the latest
genus of lampreys to evolve (Gill et al. 2003), and was the focus of several early studies
investigating steroids in early vertebrates (Kime and Rafter 1981; Larsen 1987, 1990;
Kime and Larsen 1987). One study found that immature adult river lamprey gonads of
either sex metabolized progesterone and testosterone into 15 -hydroxylated derivatives in
vitro (Kime and Rafter 1981), although there were questions regarding whether the
hydroxylation occurred at the a or [3 position (Kime and Callard 1982). Recent research
has shown that 15 a-hydroxytestosterone (lSa-T) and [So-hydroxyprogesterone (lSa-P)
are the major products of sea lamprey (Petromyzon marinus) testis in vitro, are circulated
in the plasma, and plasma concentrations increase in response to injections with
gonadotropin-releasing hormone (GnRH) in prespermiating adult males (Bryan et a1.
2003, 2004; Young et al. 2004). Additionally, it has recently been shown that 15(1-
hydroxylated steroids are produced in vitro and in vivo in the males of four other North
American lamprey species, including the silver lamprey (Ichthyomyzon unicuspis),
chestnut lamprey (I. castaneus), American brook lamprey (Lethenteron appendix), and

Pacific lamprey (Entosphenus tridentatus) (Bryan et al. submitted).

Lampreys have been considered the best living proxy for a vertebrate ancestor (Neidert et
a1. 2001), and constitute a key group for testing hypotheses regarding the timing and
evolution of the steroid-receptor system (e. g., Thornton 2001; Baker 2004). To properly
interpret the significance of lSa-hydroxylated steroids in the context of the evolution of

steroids in vertebrates, the evolution of these steroids must be investigated in the context

123

 

of lamprey phylogenetics. It has already been demonstrated that 150t-T and 150t-P are
present in the ancestral lamprey genus, Ichthyomyzon (Potter 1980; Bryan et al.
submitted). The river lamprey belongs to the latest extant genus to evolve, Lampetra
(Potter 1980; Gill et al. 2003), making it particularly important when addressing
questions regarding the commonality of a particular trait in lamprey phylogenetics. The
question as to whether 15(1- or lSB-T, or both, are produced in river lampreys has never
been resolved, and in vivo production of 15 (it-hydroxylated steroids has not previously
been investigated. Further evidence regarding the status of these unusual steroids in
Lampetra species would provide support for the theory that ISa-hydroxylated steroids are

a common feature among species within the Petromyzontidae family (holarctic lampreys).

In this study, the objectives were to 1) Confirm in vitro production of ISa-hydroxylated
steroids by mature adult river lamprey testis, 2) Determine if the in vivo production and
circulation of lSa-hydroxylated steroids changes as natural maturation occurs in male
lampreys, and 3) Determine if injections of either lamprey GnRH I or III (Sherwood et al.
1986; Sower et al. 1993) cause an increase in plasma concentrations of lSa-hydroxylated

steroids in male river lampreys.

Methods

River lampreys were captured from River Ouse near Acaster Malbis in early December
2003 and transported to the University of Durham, where they were held in flow-through
tanks at ambient temperatures and light conditions. Additionally, lampreys were

captured while nesting on the River Ure near Ripon on April 20, 2004 which were also

124

transported to the University of Durham and held at ambient temperatures and light
conditions for one day prior to sampling. Animal were anesthetized in 1:5000 MS-222
prior to handling for experiments. All chemicals were obtained from Sigma-Aldrich (St.
Louis, MO, USA), unless otherwise noted. Lamprey GnRHs were synthesized by
Bachem Peptide Company (King of Prussia, PA, USA) according to Sherwood et al.

(1986) for GnRH I and Sower et al. (1993) for GnRH III.

In vitro experiments. The in vitro experiments to examine the capacity of mature river
lamprey gonads to lS-hydroxylate steroids were carried out in April 2004. Incubations
were carried out each using pooled gonadal tissue from two lampreys, and were carried
out separately for lampreys held in captivity over the winter (“tank-held” lampreys) and
for lampreys captured on the spawning ground (“fresh-caught” lampreys). Testis
incubations were conducted as previously described (Bryan et al. 2003, 2004) using 0.5 g
testis in 10 ml L-15 media for 4 h, with 25 pCi of tritiated testosterone (T) or tritiated
progesterone (P), although 75 pCi was used for the incubation of testis from tank-held
lampreys. The incubation was stopped by freezing, and the media was thawed and
extracted as by Bryan (2003, 2004) using an activated Sep-pak (Waters Corporation,
Milford, MA, USA). The identity of the ISa-hydroxylated steroid products was
confirmed through coelution with standards on high performance liquid chromatography
(HPLC) and thin layer chromatography (TLC), coelution of acetylated products and
standard on TLC, and binding to specific antibodies raised against standards (Bryan et al.

2003, 2004). Although ISa-T and ISB-T elute less than one minute apart on HPLC, they

125

do not co-migrate on TLC, and therefore the two very similar molecules can be

distinguished from one another.

Natural changes in plasma steroid concentrations. Blood was sampled using heparinized
syringes from 20 tank-held lampreys each in December, February, and March, and from
32 lampreys in April. Blood was also sampled from 26 fresh-caught lampreys in April.
The lampreys were killed so that their sex could be determined surgically, and gonadal-
somatic index (GSI) could be recorded. The blood was centrifuged at 1000 x g for 20
min, the plasma pipetted off, and stored at -20° C. The plasma was assayed using
radioimmunoassays (RIAS) for ISa-T (Bryan et al. 2003) and ISa-P (Bryan et al. 2004).
Differences in steroid levels and GSI among months for tank-held lampreys of each sex
were analyzed using an ANOVA, followed by multiple comparison tests using a
Bonferroni adjustment if significant results were obtained. Correlations between GSI and

steroid levels were also investigated.

Effect of GnRH on plasma steroid concentrations. The GnRH-injection experiment was
performed using tank-held lampreys only. There were three treatment groups, each
consisting of eight males, and each treatment was placed in a separate tank. Lampreys
were given two serial injections, 24 h apart, of either GnRH I (100 pg/kg, Sherwood et al.
1986), GnRH III (100 pg/kg; Sower et al. 1993), or saline as a control. Blood was
sampled 6 h after the second injection, processed to obtain plasma, and assayed as
described above. The assay was further validated by pooling plasma from lampreys

treated with GnRH I and GnRH III. The plasma was extracted using a Sep-pak and

126

 

fractionated using HPLC as described above. The HPLC-fractionated plasma was then
assayed for both lSa-T and ISa-P, and immunoreactivity was compared to the known
elution times of the steroids. Differences in steroid levels among treatment groups were
investigated using ANOVA, and Bonferroni adjustments for multiple comparison tests
were used to determine differences between specific groups if significant results were

obtained.

Results

In vitro experiments. Males produced both 150t-T and ISa-P from 3H-T and 3H-P in vitro,
but 3H-P was converted into additional products as well (Fig. 21). The identity of the
ISa-hydroxylated steroids was confirmed through coelution with 150t-T or ISa-P on
HPLC and TLC, coelution of acetylated in vitro products and acetylated standard on TLC,

and binding to specific antibodies raised against lSa-T.

Natural changes in plasma steroid concentrations. Tank-held males experienced

changes in plasma steroid levels from the time of their migration to the time that
spawning takes place (Fig. 22). 15 a-T levels in males did not change significantly in the
tank-held lampreys (ANOVA, d.f. = 3, 47, P > 0.0500) and the fresh-caught males had
significantly less lSa-T than tank-held lampreys in April (t-test, d.f. = 25, P = 0.0144).
ISa-P levels in tank-held males changed significantly (ANOVA, d.f. = 3, 47, P < 0.0001),
and plasma levels of 15(1-P were lower in December than in February (P = 0.0050),
March (P = 0.0030), and April (P < 0.0001). Fresh-caught males had less lSa-P than

tank-held males in April (t-test, d.f. = 25, P = 0.0157). Average tank-held male GSI

127

changed significantly among months (Fig. 23; ANOVA, d.f. = 3,47, P < 0.0001).
Average male GSI was higher in March than in December (P < 0.0001) and in February
(P = 0.0020), and was lower in April than in December (P = 0.0440), February (P =
0.0060) or March (P < 0.0001). GSI did not have significant correlations with either

15(1-T or 150t-P.

128

 

  

 

 

15a-T T
l 1
Q
40 - 31.
it
C :2
.2 30 « .
in!
.2
“a
a
g 20 , + Tank-held
° --0- Fresh-caught
10 -
0
20 30 40 50 60 70
HPLC fraction
15a-P P
50 - \i/ \i/
-O— Tank-held e
--o- Fresh-caught
40 ~
:
.2 sol
“
.2
p
g ..
.\° 2° ‘

 

 

 

 

HPLC fraction

Figure 21: Results investigating in vitro and in vivo production of ISa-hydroxylated
steroids in male river lampreys. The x-axis is HPLC fraction and the y-axis is percent of
total radioactivity. Arrows indicate the known elution points of 15(1-P, progesterone (P),

lSa-T, and testosterone (T).

129

m 15a-T
[:3 15a-P

7%
f
19

 

 

 

 

 

 

 

 

 

 

l—m—fi
Feb. Mar. Apr. (Cap.)Apr. (Wild)

Date of sample

Figure 22: Changes in plasma steroid levels between the time of upstream migration and
spawning in river lampreys. Plasma was obtained and assayed for 150-
hydroxytestosterone (lSa-T) and ISa-hydroxyprogesterone (ISa-P). The sex of the
animals was confirmed surgically. The first four bars represent lampreys captured during
their upstream migration and held at ambient light and temperature conditions, and the
last bar represents lampreys that over-wintered in streams, and were captured while
nesting or spawning. The error bars represent one standard error of the mean.

0.09 '-
0.08 ‘ é
0.07 r

0.06 '1

0.05 - q)

0.04 -

GSI
r04

0.03 ‘

 

 

0.02 . . . .
Dec Feb Mar Apr
Sample month

Figure 23: Mean GSI of tank-held male lampreys in different months. The error bars
represent the standard error of the mean.

130

Effect of GnRH on plasma steroid concentrations. Neither type of GnRH had a
significant effect on circulatory lSa-T levels. Control 150t-T levels were (mean d:
standard error) 0.13 d: 0.03 ng/ml. However, both types of GnRH had significant effects
on circulatory 1501-P levels in males (ANOVA, d.f. = 2,20, P = 0.0010), but there was no
difference between the effects of the two different types of GnRH. Control levels of 1501-
P in males were 1.86 i 0.17 ng/ml, levels in lamprey treated with GnRH I were 3.72 :t
0.44 ng/ml, and levels in lampreys treated with GnRH III were 4.48 i 0.61 ng/ml (Fig.

24).

 

 

 

 

 

 

 

 

 

 

 

7 l
6 l
A 51 l
'E
‘ 4 .I
g l
t. a -
In
1-
2 '1 T
1 .
o I I I I
control GnRH I GnRH III
Treatment group

Figure 24: lSa-Hydroxyprogesterone (l Sa-P) plasma concentrations of male lampreys
given two serial injections, 24 h apart of GnRH (100 pg/kg). Blood was sampled 6 h
after the second injection. GnRH I and GnRH 111 both elicited significantly higher
plasma levels of ISa-P than saline injections in male lampreys. Neither type of GnRH
significantly increased plasma levels of ISa-hydroxytestosterone (ISa-T) in male
lampreys.

131

3 - 15a-P P

21

ir-150t-P (nglml)

 

 

25 30 35 40 45 50 55 60 65

0.5 -
15a-T T

0.4 . j j

0.3 .

0.2 ‘

lr-1 5a-T (nglml)

0.1 a

(,0. ‘I A u A

25 30 35 40 45 50 55 60 65 70
HPLC Fraction

 

Figure 25: Immunoreactivity (ir) to antibodies raised against lSa-hydroxyprogesterone
( lSa-P) and lSa-hydroxytestosterone (lSa-T) in HPLC-fractionated river lamprey
plasma. Plasmas from lampreys injected with GnRH I or GnRH III were pooled,
extracted, fractionated using HPLC, and the fractions assayed using RIA. Arrows
indicate the known elution points of lSa-P, progesterone (P), 150t-T, and testosterone (T).

132

In HPLC-fractionated male river lamprey plasma, there were five peaks of ir-150t-P (Fig.
25). The largest of these peaks coeluted with 15(1-P, and was 49.8% of the total
immunoreactivy. Of the remaining peaks, one coeluted with P (64 min), and the others
did not correspond with available standards, and were at 32, 37, and 46-47 min. There
was one major peak of ir-lSa-T, which coeluted with lSa-T and was 50.7% of the total

immunoreactivity.

Discussion

Male European river lampreys appear to produce lSa-hydroxylated steroids in the gonads
and circulate them in the plasma. Males produced both ISa-T and ISa-P in vitro,
although other steroids were produced, and relatively large quantities of P were left
unconverted. Additionally, although the putative lSa-T produced in vitro in males
coeluted with standard on TLC in both its unaltered and acetylated forms, and bound
strongly to antibodies raised again 1501-T, it also exhibited binding to antibodies raised
against ISB-T. The binding to antisera raised against lSB-T may be due to contamination
of the standard lSB-T which was used in raising antibodies with 150t-T (Bryan et al.
2003). In sea lampreys, it was found that the conversion of P to three other steroids, one
of which was identified as 15(1-P, changed as the reproductive season progressed, with
the synthesis of lSa-P being greatest earlier in the reproductive season (Bryan et al.
2004). River lampreys may exhibit a similar phenomenon, and this may also explain the
discrepancies in the results between this study and a previous one (Kime and Rafter 1981)
which used river lampreys immediately after being captured during their upstream

migration.

133

River lampreys have a semelparous, anadromous life cycle in which larvae are filter
feeders which live in stream sediment, followed by a parasitic phase which lives in the
ocean, and adults migrate to streams to spawn in gravel (Maitland 1980). The upstream
spawning migration takes place in the fall, and lampreys overwinter in a fasting state in
streams before spawning in spring (Maitland 1980). Levels of steroids changed in males
during the time between their upstream migration in December and the time at which
spawning occurs naturally in April. Plasma concentrations of ISa-P increased as the
season progressed, but the significant increase occurred between December and February.
Again, more research is necessary to determine the function of lSa-P, and further
research may determine if it regulates final maturation in lampreys , as progestagens are
known to do for many teleost fish species (review: Miura and Miura 2001). Levels of

ISa-T did not change throughout this period.

The lower steroid levels found in fresh-caught lampreys in April are likely due to the
stress of capture and transport, as plasma was sampled the day after they were removed
from the river. Laboratory conditions for tank-held lampreys mimicked the natural
photoperiod and temperatures that the fresh-caught lampreys would encounter. However,

lampreys held in captivity since December were acclimated to being held in a tank.

Despite statistically significant changes in plasma steroid levels at different times of

sampling, it is still unknown how, and if, these steroids function within the HPG axis.

lSa-T levels did not change in response to either type of GnRH in males or females. If

134

lSa-T does not have a physiological function near the time at which sexual maturity
occurs, levels of this steroid may not be affected by GnRH. As maturation progresses,
many teleost fish exhibit a shift in steroidogenesis from androgens (C19 steroids) to
progestagens (C21 steroids) (Scott et al. 1983; Barry et al. 1990). However, at this time,
it has not yet been determined whether lSa-T is a functional hormone, and its lack of
response to GnRH may also suggest a lack of function. Additional experiments using
lampreys at earlier life stages would be useful in determining whether the lack of

response to GnRH seen in this study is stage-specific in river lampreys.

In contrast to its effect on lSa-T, both types of GnRH increase circulatory levels of lSa-P.
The data obtained from this experiment are particularly puzzling, considering the data
obtained from non-injected fish used to examine changes in steroid levels during the
overwintering period. Tank-held males used to examine seasonal differences (no
injections) had higher plasma concentrations of ISa-P than control males, and similar

levels to GnRH-injected fish.

The results from the experiments using GnRH are different than those obtained using
other species of lampreys. Using the same injection protocol as in this study, at 8 h after
the second injection, male sea lampreys (Petromyzon marinas) exhibit a 2-5 fold mean
increase in lSa-T levels (Young et al. 2004), and a 36 fold mean increase in ISa-P levels
(Bryan et al. 2004). The disparity may be due to life history differences and associated
physiological differences, as sea lampreys migrate upstream in the spring directly before

spawning (Manion and Hanson 1980). Additionally, using a different injection protocol

135

in which lampreys were given one injection of GnRH and plasma sampled 24 h later,
neither type of GnRH caused increased levels of lSa-T or ISa-P in male silver lampreys
(Ichthyomyzon unicuspis) near spawning and only GnRH III caused increased levels of
ISa-T and lSa-P in male Pacific lampreys (Entosphenus tridentatus) several months

prior to spawning (Bryan et al. submitted).

Of the lampreys species thus far investigated, the testes of all of them have the capacity
to produce lSa-hydroxylated steroids in vitro, and immunoreactive lSa-hydroxylated
steroids have been detected in the plasma. Although the synthetic pathway to produce
ISa-hydroxylated steroids seems functional in all male lampreys studied, the actual
patterns and regulation of these synthetic pathways appears to differ among species. In
particular, there appears to be vast differences among species in how GnRH treatment
affects circulating levels of lSa-hydroxylated steroids. The differences in responses
cannot be explained by phylogenetics, as sea lampreys and silver lampreys belong to a
monophyletic group, and Pacific lampreys and river lampreys evolved later from a
common ancestor (Gill et al. 2003). There are differences in life history in these species,
as river and Pacific lampreys cease feeding and migrate upstream in the fall and
overwinter in streams before spawning (Maitland 1980; Beamish 1980), while silver and
sea lampreys migrate upstream in the spring and have a much short fasting period
(Manion and Hanson 1980). Additionally, the feeding ecology of each of these species of
lampreys differs as well, and based on dentition, silver and sea lampreys ingest host
blood, while Pacific and river lampreys ingest host body tissues (Potter and Hillard 1987)

although it appears that the pacific lamprey attacks ventrally and prefers to ingest body

136

fluids (Beamish 1980). The differences in results may also be, explained by differences

in experimental protocol, or by the maturation stage at which the experiments took place.

Further research is needed to elucidate the function of the ISa-hydroxylated steroids in

all lamprey species, and to better understand how life history differences among lamprey

species affect reproductive endocrinology.

137

Acknowledgements

Funding for this research was provided by the National Science Foundation #INT-
0336929. We thank Alistair Cook from CEFAS for his assistance with the GnRH
experiments, Brian Morland for help providing spawning lampreys, and Jesse Semeyn

from MSU for his assistance with RIA.

138

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adrenal and sex steroid receptors. Mol. Cell. Endocrinol. 215, 55-62.

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river lamprey, Lampetrafluviatilis, in vitro. Gen. Comp. Endocrinol. 44, 69-76.

Kime D.E., Larsen LC. 1987. Effect of gonadectomy and hypophysectomy on plasma

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Endocrinol. 68, 197-201.

Larsen L.O. 1990. The role of external and internal factors during the phase of
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fiom the upper three Great Lakes. Can. J. Fish. Aquat. Sci. 37, 1635-1640.

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(Petromyzontidae). J. Applied Ichthyol. 13, 143-148.

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Sower SA. 1990. Neuroendocrine control of reproduction in lampreys. Fish Physiol.
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Sower S.A., Chiang Y., Lovas S., Conlon J .M. 1993. Primary structure and biological
activity of a third gonadotropin-releasing hormone from lamprey brain.
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estrogen receptor by ligand exploitation and serial genome expansions. PNAS 98,
5671-5676.

Young B.A., Bryan M.B., Sower S.A., Scott A.P., Li W. 2004. ISot-hydroxytestosterone

induction by GnRH-I and GnRH-III in Atlantic and Great Lakes sea lamprey
(Petromyzon marinus L.). Gen. Comp. Endocrinol. 136, 276-281.

141

APPENDIX

Young B.A., Bryan M.B., Sower S.A., Scott A.P., Li W. 2004. 1501-
Hydroxytestosterone induction by GnRH-I and GnRH-III in Atlantic and Great Lakes sea

lamprey (Petromyzon marinus L.). General and Comparative Endocrinology 136, 276-
281.

142

lSa—Hydroxytestosterone induction by GnRH I and GnRH III in Atlantic and Great
Lakes sea lamprey (Petromyzon marinas L.)

Bradley A. Young“, Mara B. Bryan*, Stacia A. Sower:r Alexander P. Scotti, and
Weiming Li""l

* Department of Fisheries and Wildlife, Rm. 13, Natural Resources Building, Michigan
State University, East Lansing, MI 48824-1222, USA; T Department of Biochemistry and
Molecular Biology, Biological Science Center, 46 College Road, University of New
Hampshire, Durham, NH 03824, USA; I The Centre for Environment, Fisheries, and
Aquaculture Science, Barrack Road, Dorset DT4 8UB, UK

Running Title: lSa-Hydroxytestosterone induction in sea lamprey

Corresponding Author:

Weiming Li

Department of Fisheries and Wildlife
Rm. 13, Natural Resources Building
Michigan State University

East Lansing, MI 48824-1222, USA

Email: liweim@msu.edu
Tel: (517) 353-9837
Fax: (517) 432-1699

143

Abstract
The sea lamprey (Petromyzon marinus L.) represents one of the two most ancient classes

of vertebrates and possesses a functional hypothalamus-pituitary—gonadal axis. However,
the presence and functionality of androgens in the sea lamprey remain elusive. Recently,
ISa-hydroxytestosterone (lSa-T) has been found in sea lamprey gonads and blood
plasma. In this study we examined changes of circulatory concentrations of lSot-T in
response to gonadotropin releasing hormone (GnRH) treatments. Plasma concentrations
of 150t-T in sea lamprey increased 2 to 5 times for all GnRH-injected sea lamprey
compared to controls (P < 0.0001). However, there were no differences among responses
1) to the two forms of GnRH (lamprey GnRH I or lamprey GnRH III), 2) to the doses
delivered (50, 100, or 200 pg/kg), or 3) between post-injection sample intervals (8 or 24
h). Between lampreys from the Atlantic Ocean and Great Lakes sites, two of seven
GnRH form and dosage comparisons showed between-site differences, but were not
believed to represent an overall between-site difference. These are the first data to show

a response of a C19 steroid to GnRH stimulation in sea lamprey.

144

Introduction

The sea lamprey (Petromyzon marinus L.) is among the most primitive of extant
vertebrates and has a complex life cycle (Hardisty and Potter 1971). Sea lampreys begin
their lives in freshwater as blind, filter-feeding ammocoetes (larvae). After three to seven
years, metamorphosis occurs and the ammocoetes become sexually immature, free-
swimming juveniles that migrate to the sea (Atlantic Ocean) or lakes (Great Lakes).
During an approximate lS-month parasitic phase, gametogenesis progresses and
spermatogonia proliferate and develop into primary and secondary spermatocytes in
males. Alter migration to freshwater streams, both sexes undergo final reproductive
maturation. The semelparous life cycle of the sea lamprey ends soon after spawning in
streams. The sea lamprey is the only jawless fish in which the hypothalamus-pituitary-
gonadal (HPG) axis has been established (Sower 1998; Sower and Kawauchi 2001).
Therefore, further knowledge regarding the reproductive physiology of sea lampreys can
aid in understanding the evolution of endocrine controls for reproduction seen in more

modern vertebrates.

The neuroendocrine components of the reproductive endocrine system are highly
conserved among vertebrates, including lampreys (Thornton and DeSalle 2000; Sower
1998; Sower and Kawauchi 2001; Thornton 2001). However, the roles of gonadal
steroids appear to differ in lampreys. Several groups have detected very low
concentrations of immunoreactive testosterone in plasma of male and female sea
lampreys, but have been unable to demonstrate that it increases in response to injections

of lamprey gonadotropin releasing hormone (GnRH) and heterologous gonadotropin(s),

145

or that its circulatory concentrations and profiles are sexually dimorphic (Katz et al.
1982; Sower et al. 1985a, 1985b; Linville et al. 1987). In addition, Ho et al. (1987)
demonstrated estrogen binding, but not androgen binding to receptors in lamprey testis.
These studies were later supported by a study that identified the cDNA that appeared to
encode a putative estrogen receptor, but did not identify any cDNA sequences that are
homologous to typical vertebrate androgen receptors in sea lampreys (Thornton 2001).
these findings support the hypothesis (Sower 1990, 1998) that cyclostomes may not use

androgens as male reproductive hormones.

One other possible explanation for the absence of experimental evidence for typical
androgens and their corresponding receptors is that lamprey androgens are structurally
different from those identified in other vertebrates. Kime and Rafter (1981) and Kime
and Callard (1982) showed that the testis of Lampetra fluviatilis and Petromyzon marinus
have the capacity to hydroxylate testosterone at the C15 position, forming 1501-
hydroxytestosterone (lSa-T). This has recently been confirmed by two studies. Lowartz
et a1. (2003) found steroids produced in vivo and in vitro by sea lampreys that have
elution times corresponding to ISa-hydroxylated steroids when analyzed with High
Performance Liquid Chromatography (HPLC). Bryan et al. (2003) used HPLC, Thin
Layer Chromatography (TLC), and microchemical analysis to further show that sea
lampreys produce lSa-T in vitro. Bryan et al. (2003) also developed a
radioimmunoassay (RIA) for ISa-T and showed this steroid to be present in the blood
plasma of male sea lampreys (and to a lesser extent in females). Despite the

identification of lSa-T in sea lampreys, the function of this steroid has yet to be

146

characterized.

This study was designed to determine a potential endocrine role of 1501-T in the sea
lamprey through induction by GnRH, thus showing this sex steroid to be a possible
component of the well-established and functional hypothalamus-pituitary-gonadal (HPG)
axis of the sea lamprey (Sower and Kawauchi 2001). In particular, the effects of lamprey
GnRH I (Sherwood et al. 1986) and GnRH III (Sower et al. 1993) on lSa-T plasma
concentrations were measured. Using adult male sea lampreys, an experiment was
designed to test: 1) the effects of the administration at different doses and at different
times post-treatment of exogenous lamprey GnRH I and GnRH III on ISa-T plasma
concentrations; and 2) to determine if there was a difference in response between adult

prespermiating male sea lampreys from the Great Lakes or Atlantic Ocean.

Methods

Pre-spermiating, adult male sea lampreys were collected during late-spring (May)
spawning migrations. Anadromous lampreys were trapped at the Cocheco River fish
ladder in Dover, New Hampshire, USA. Great Lakes lampreys were trapped at the
mouths of two Lake Huron tributaries, the AuGres and AuSable rivers in Michigan, USA.
Atlantic Ocean lampreys were held at the University of New Hampshire (Durham, NH)
and Great Lakes lampreys were held at the Hammond Bay Biological Station (USGS-
BRD, Millersville, MI) in tanks containing approximately 160 L of continuous-flow
water for at least two days prior to treatment applications. Water at the New Hampshire

site came from the Oyster River while the Michigan site used Lake Huron water.

147

Temperature during acclimation and experimental periods was maintained at 16 °C (:1
°C) for both sites. Lamprey were weighed and measured for use in subsequent dosage

calculations.

Lamprey GnRH I and lamprey GnRH III were synthesized by American Peptide
Company (Sunnyvale, CA) and dissolved in 0.6% saline less than 30 minutes prior to
administration. Each site used seven treatments, twelve lampreys per treatment, with two
timed injections, and two timed bleedings as done previously (Sower 1989; Deragon and
Sower 1994; Gazourian et al. 2000). Each group of 12 lamprey were injected
intraperitoneally with either 0.6% saline (control), lamprey GnRH I (50, 100, or 200
pg/kg body weight), or lamprey GnRH III (50, 100, or 200 pg/kg body weight). The
two injections, 48 h apart, were administered beginning at 8:00 am. Blood samples (0.5-
1.0 ml) were collected 8 h and 24 h after the second set of injections by cardiac puncture
using heparinized syringes. After centrifugation of blood samples, plasma was collected
and stored at -80 °C until analyzed for 150t-T by RIA according to Bryan et al. (2003).
The RIA for lSa-T was conducted using raw plasma. The antibody raised against lSa-T
was used at a dilution of 1:100,000 and radiolabel was dispensed so that there were 7,000
dpm per tube. In the absence of standard, the antibody bound approximately 50% of the
available radiolabel. The sensitivity of the RIA was 39 pg/ml plasma and had a

detectable range from 2 - 500 pg/tube.

The reliability of the 150-T RIA, when applied to plasma from GnRH-injected lampreys

was validated in three ways. First, 5 ml of pooled plasma from GnRH-treated lampreys

148

were extracted using a solid phase extraction cartridge (Sep-Pak C18; Waters, Milford,
MA), fractionated using HPLC (Bryan et al. 2003), and 20 p1 of each fraction then
assayed for ISa-T using RIA. This was done in order to confirm that immunoreactivity
was restricted to the expected elution position of ISa-T and that production of no new
immunoreactive compounds had resulted from GnRH induction. Second, dilutions of
pooled plasma from GnRH-treated lampreys were combined with labeled 150t-T in the
absence of antibody. This was done to confirm the absence of plasma binding proteins
(which if they were present, might interfere with the RIA). Third, standard 15a-T at a
range of dilutions was added to plasma from GnRH-injected lamprey and assayed at
volumes of 25 pl, 50 pl, and 100 pl (total volume brought to 100 pl with assay buffer).

This was done to establish parallelism within the assay.

Results were analyzed at multiple treatment levels. Violations of normality and variance
equality precluded the use of a factorial analysis of variance (ANOVA). For comparisons
among several treatment groups, Kruskal-Wallis (K-W) tests were used. T-tests or
Wilcoxon signed-ranked tests (W) were used for simple group comparisons. There were
14 interval comparisons (7 treatments x 2 sites) between the 8 h and 24 h samples. If
those comparisons proved not significant, then the two intervals were pooled. Second,
comparisons of the three dosage levels for GnRH I and GnRH III were made. Third,
differences between the two forms of lamprey GnRH were evaluated by dose and site.

Finally, between-site differences were assessed by GnRH treatment and dose.

149

Results

During the course of the experiment, 24 of the 168 lampreys died. Mortalities among
treatments appeared random with the highest number (50%) seen in a control treatment.
Lamprey size differed between the sites where Great Lakes sea lampreys had a mean
length of 509 mm and mean weight of 243 g while Atlantic sea lampreys had an

approximate mean length of 700 mm and an approximate mean weight of 800 g.

The RIA of HPLC fractions from GnRH-injected male plasma revealed a single peak of
immunoreactivity corresponding to the known elution position of ISot-T (Figure 26). The
test for binding proteins in the plasma was negative. The test of parallelism returned a

line with a slope equal to the known values and an r2 = 0.86.

1000 -
900 r
800 i
700 r
600 r
500 r
400 r
300 r
200 r
100 -

O . 1 . . a . . . .
20 24 28 32 36 40 44 48 52 56 60 64 68 72
HPLC fraction

e<—-150t-T

 

 

Immunoreactive lSor—T (pg/fraction)

 

 

 

fl #7 I

Figure 26: Radioimmunoassay of HPLC-fractionated plasma from GnRH-injected male
sea lampreys. The elution point of ISa-hydoxytestosterone (lSor-T) standard is indicated
with an arrow.

150

Plasma analyzed using RIA yielded 1501-T concentrations ranging from 0.07 to 4.52
ng/ml and one extreme value of 7.97 ng/ml that was excluded from analyses. Average
standard reference curve error was 13% at the midpoint and 29% at the low end. The
data were not normally distributed (Shapiro-Wilk, P < 0.0001) at both sites and variation

among the Atlantic lamprey data (0'2 = 1.20) was more than double that of the Great

Lakes site ((52 = 0.55). Treatment subset groupings of data did favor some parametric

comparison tests, but non-parametric tests were necessarily used in most cases.

The 14 interval comparison tests showed three significant differences between the two
sample times (8 and 24 h). This proportionally small number of differences was believed
to not represent a true interval difference and led to the pooling of these two time
intervals for further analyses. There was also no evidence of dose response differences
within Great Lakes-GnRH I (P = 0.82), Great Lakes-GnRH III (P = 0.58), Atlantic-
GnRH I (P = 0.11), and Atlantic-GnRH III (P = 0.19) groups. Additionally, no
significant difference in lSa-T concentrations was found between lampreys injected with
GnRH I or GnRH III at the Great Lakes site (P = 0.93), nor at the Atlantic Site (P =

0.16).

Only two of seven GnRH form and dosage specific treatment comparisons differed
between the two sites. The GnRH III, 50 pg/kg treatment differed between sites (W, P =
0.018) where median ISa-T plasma concentrations were 2.04 ng/ml at the Atlantic site
and 1.26 ng/ml at the Great Lakes site. The control groups (saline-injected) also differed

(W, P = 0.006) where median lSor-T plasma concentrations for Atlantic sea lampreys

151

were 0.44 ng/ml and 0.29 ng/ml for Great Lakes sea lampreys. The ratios of lSa-T

concentrations in Great Lakes versus Atlantic sea lampreys were similar for both GnRH-

injected lampreys (1:1.31) and control group lampreys (1 : 1.52).

   

Great Lakes

:1 8Hours

w///////////////////////////////////xm

 

3.0 -

2.5 1 24 Hours

 

I

I

I

7/////////////////n

I

I

mm I

M I
-2

GEES 055605on06382

200

00

1

200 50
Lamprey GnRH III

100

0.6% Saline 50

Lamprey GnRH I

Control

Figure 27: Differences in ISa-hydroxytestosterone blood plasma concentrations for

male sea lampreys per site, GnRH form, dosage, and time interval. The X-axis labels

indicate the GnRH form and dosage in pg/kg of lamprey body weight. Bars represent

mean treatment values and error bars show mean standard error.

152

The effect of GnRH injections (regardless of dose or form) was evaluated by comparing
ISa-T concentrations between all GnRH-injected lampreys of each site to their
corresponding saline-injected control group. Because the Great Lakes control group
showed some differences between time intervals, the comparisons were made per interval
and with combined interval data. Large differences in sample size from pooling data led
to GnRH-specific and dosage-specific comparisons. At both sites and for every level of
classification (interval, dose, and GnRH form), GnRH-injected lampreys always had

significantly higher (K-W, P < 0.0001) plasma concentrations of ISa-T than control

groups (Figure 27).

Discussion

The results showed that both GnRH I and GnRH III elicited increases in ISa-T
production and release in the blood plasma of adult male sea lampreys. GnRH-injected
sea lampreys had 2 to 5 times greater 150t-T concentrations than their respective controls.
The identity and presence of ISa-T in sea lamprey blood plasma was only recently
confirmed and shown to be present at higher concentrations than immunoreactive
testosterone (Bryan et al. 2003; Lowartz et al. 2003). This study now shows that ISa-T
has a physiological response to GnRH and may be part of the HPG axis as induced by

GTH.

The response of ISa-T further adds to the growing understanding of the unique endocrine
system of the sea lamprey. To date, research has demonstrated the effects of

hypothalamus and pituitary hormones on 17B-estradiol and progesterone production in

153

sea lampreys (Sower and Kawauchi 2001). GnRH I and GnRH III directly stimulate the
pituitary and increase steroidogenesis (Gazourian et al. 2000). In vitro receptor
localization studies have identified areas of the pituitary with an affinity for GnRH I and
GnRH 111 (Knox et al. 1994) and estrogen receptor sites in the testis (Ho et al. 1987). In
male sea lampreys, immunoreactive 17B-estradiol (E2) has been found in blood plasma
(Sower et al. 1983; Sower 1989; Katz et al. 1982), whose concentrations increase in
response to injections of lamprey GnRH I, GnRH III, and GnRH analogs (Sower et al.
1983, 1985b, 1993; Sower 1989; Deragon and Sower 1994; Gazourian et al. 1997).
Additionally, receptors for E2 have been found in the testis (Ho et al. 1987).
Concentrations of lSa-T showed the same proportional (injected versus control)
increased responses to GnRH I and GnRH III as E2 responses have shown (Young,
unpubl. data). Expected receptor gene sequences for androgens were not found in the
testis of lamprey (Thornton 2001), but the author indicated that does not exclude the
possibility that very different receptor sequences exist for androgens, including lSa-T, in
sea lampreys. Finding this response of ISa-T now requires further research to reveal
whether it, like E2, has receptor binding sites in the testis, thus supporting the hypothesis
that C19 steroids may have an androgenic role in the sexual development and maturation

processes of sea lampreys.

A possible explanation for the absence of change in ISa-T concentrations between the 8
h and 24 h sampling times may be that the duration selected was too short to observe the
progression of increase and was not long enough to observe the pending decrease.

Previous studies have shown that final maturation was accelerated by one or more

154

injections of GnRH (Sower 1989). The first injection is believed to activate the system,
making it more sensitive to further injections. Sower (1989) shows how a single GnRH
injection maintains the concentration of E2 and progesterone (P) after 24 h, whereas after
two successive GnRH injections, concentrations began to decrease after 24 h. The results
from this study appear to indicate that for ISa-T responses, the two successive GnRH
injections did not push the lampreys past the initial activation stage, thus the samples at

the 8 h and 24 h sample times showed no differences in concentration.

The absence of dose-dependent responses indicates that the minimum GnRH doses used
in this study were sufficient to induce the maximum 150t-T response in sea lamprey.
Deragon and Sower (1994), using four successive injections, found E2 concentrations to
increase within 4 h, yet found no difference in responses between 100 and 200 pg/kg
doses Gazourian et al. (1997), using four successive injections, found that
concentrations of both E2 and P rose after 4 h and began to descend after 24 h. Gazourian
et al. (2000), using a single injection and doses of 50 and 100 pg/kg, found similar
elevated E2 and P responses after 4 h, but a less pronounced decline after 24 h and that
the 50 pg/kg dosage elicited lower responses in some instances. All studies found that
higher temperatures generally produced higher concentrations of steroids. Based on the
experimental design of these previous studies, the injection regime, sampling intervals,
and temperature used in this study were expected to elicit differential ISa-T
concentration responses between intervals and among doses. The fact that differences
were not seen in these treatments as were seen for E2 and P in other studies, suggests that

the production of lSa-T may be regulated differently than the production of estrogens

155

and progestogens

Within the range of doses used in this study, lamprey GnRH I and III were equipotent in
increasing concentrations of 150t-T. Sower et al. (1993) first identified GnRH III and
found that it elicited E2 responses similar to those of GnRH 1. Other studies have also
shown that GnRH I and GnRH III are equally effective in elevating E2 and P
concentrations in sea lamprey (Sower 1998). Gazourian et al. (1997) showed that GnRH
I and GnRH III were equipotent in stimulating steroidogenesis and inducing ovulation in
females. Only Deragon and Sower (1994) found different responses to the two GnRH
forms where GnRH III in males was more potent than GnRH I in its ability to induce
spermiation and increase the concentrations of E2 and P. The elevated concentrations of
lSa-T seen in response to both GnRH I and GnRH III indicate that, at the doses
examined, the different GnRH forms appear to have equal effects on this steroid and are

similar to the responses of estrogens and progestogens.

The two GnRH form and dose-specific comparisons that differed between sites are not
believed to represent an overall between-site difference. There was less than 39%
difference in median plasma concentrations between sites for both of these two
comparisons. This is low when compared to the over 72% difference in median plasma
concentration between GnRH-treated lampreys and their respective controls. Although
lamprey size and environmental conditions do differ between sites, the majority of the
data support the conclusion that there is minimal if any difference in lSor-T plasma

concentrations between the two sites.

156

The ISa-hydroxylated form of testosterone found in sea lampreys is unconventional and
may be indicative of more specialized functions necessary in this ancient vertebrate.
There have been ISa-hydroxylated steroids found in other organisms, but not as sex
hormones (Levy et al. 1965; Giannopoulos et al. 1970; Brown et al. 1979). A possible
explanation for the use of lSa-T by lampreys is the assumption that a hematophagous
parasite needs different forms of hormones than its hosts to avoid interaction with
exogenous hormones. However, because sea lamprey only have a short parasitic phase
while the majority of their life cycle is spent as larvae with immature gonads, the reason
for needing hydroxylated testosterone and its specific functions are not yet known.
Though the origins and function of lSa-T may be in question, its placement in the HPG
axis and response to lamprey GnRH add significant information toward the further

understanding of the reproductive endocrinology of this ancient fish.

157

Acknowledgements

Funding for this study was provided by the Great Lakes Protection Fund and the Great
Lakes Fishery Commission to Weiming Li and by NSF #0090852 to Stacia Sower. We
thank Hammond Bay Biological Station (USGS-BRD) for use of their facilities and the
USFWS for collecting the Great Lakes lamprey. We also thank those who assisted with
conducting the experiments: R. Seymour, J. Glenn, K. Kucher, J. Semeyn, M. Stewart, C.

Boum, J, Gleico, J. Sanford, and E. Violette.

158

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