REGULATION OF TESTOSTERONE PRODUCTION

BY CYTOKINES iN THE GOLDFISH

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

BY

ANDREA LEANNE LISTER

In partial hl filment of requirements

for the degree of

Master of Science

February, 200 1

Q Andrea L. Lister, 200 1

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REGULATION OF TESTOSTERONE PRODUCTION BY CYTOKINES LN THE GOLDFISH

Andrea Lister University of Guelph, 2001

Advisor: Dr. G. Van Der Kraak

A series of in vivo and in vitro approaches were used to investigate the regulation

of testis function by immune-derived factors in the goldfish. Administration of

lipopolysaccharide (LPS) in vivo lead to a signiticant decrease in plasma testosterone (T)

levels of goldfish exhibiting a Iow gonadosomatic index (GSI) value. The munne

cytokines, tumor necrosis factor a (TNFa) and interleukin- 1 P (IL-1 P), LPS, and teleost

macrophage conditioned media (MCM) inhibited hwnan chorionic gonadotropin ( K G ) -

stimulated T production by goldfish testis pieces in vitro. Basal T levels were affected

more variably by the compounds. The predominant site of activity of TNFa in the

gonadotropin-stimulated steroid biosynthetic pathway appears to be post-CAMP

generation but prior to the conversion of pregnenolone to other sterol precursors. TNF-

and IL- 1 -1ike peptides were immunologically detectable in LPS-stirnulated RTS L I cell

culture supemaiants but the same mammalian antibodies failed to ablate the activity of

the MCM on T levels in vitro or detect RIF- and IL-1-like peptides in the tissues of male

go Id fish. Collectively, these studies provide support for the involvement of cytokines and

possibly other immune-derived factors in the regulation of teleost testicular

steroidogenesis.

ACKNOWLEDGMENTS

At al1 points throughout this project, my advisor Dr. Glen Van Der Kraak has

provided incredible support and guidance that has been invaluable. The opportunity to

leam from someone so patient and undentanding is a privilege. The advice given to me

by my enthusiastic cornmittee members, Dr. Niels Bols and Dr. Tony Hayes, is also

grea tly appreciated.

Many parts of this project would not have been possible if i t weren't for the

generous donation of the RTS 1 I cell line by Dr. Bols and the excellent lessons in ce11

culture techniques by John Bmbacher. 1 am indebted to Sue Biddiscombe, who for the

first year of this project (and many days dunng my undergraduate projects), taught me

valuable lessons in life and lab that continuously payoff. And frankly, many thanks go to

the extended gang at CCIW (Pete, Witt, Ger ... etc.) for the wondefil, sloppy times out of

the lab.

The Van Der Kraak lab is full of' exceptional people. Many thanks to al1 that have

passed through and made my time here mernorable: Katie, Kelly, Mel, Vikki, Jacquie,

Ken*, Tony, Derek, Noreen, Andy ... And who could forget the generations of Primordial

Ooze and Clobster players. And thanks to rnany others, like Marc (for the çtats chats),

Glenn (for the DR), Jason and Nat ... etc.

Finally, fhends back home and family have been there for me. A special thanks to

my parents who have never tired of asking about the fish and who have surviveci several

fish tours and to Paul, for enduring countless moods at al1 houn. And to Bronze, thanks

for the companionship in and out of the lab.

TABLE OF CONTENTS

................................................................................................................ Acknowledgments i

. * Table of Contents ................................................................................................................. 11

... List of Table ............................................................................................................................ 111

List of Figures ......................................................................................................................... iv

...................................................................................... CHAPTER 1: General Introduction 1

CHAPTER 2: In Vitro Modulation of Goldfish Testicular Testosterone Production by

Tumor Necrosis Factor u, Interleukin-1 P, and Macrophage-Derived Products ............ 12

INTRODUCTION ..................................................................................................... 12

MATERIALS AVD METHODS .............................................................................. 1 5

................................................................................................................... RESULTS 19

............................................................................................................. DISCUSSION 39

CHAPTER 3: Modulation of Testosterone Production in Male Goldfish and Induction

of Cytokine-like Molecules in a Trout Macrophage Cell Line by the Immune Stimulant,

......................................................................................................................................... LPS 46

lNTRODUCTION .................................... ,.... ......................................................... .46

................... MATERLALS AiVD METHODS ., ....................................................... -48

RESULTS ........................... .. ..................................................................................... 53

............................................................................................................ DISCUSSION 65

............................................. CHAPTER 4: General Discussion .................................... ,,.,.. 72

REFERENCES ..................................................................................................................... 77

List of Tables

Table 3.1. A description of the average GSI ranges of male goldfish from Fig. 3.3. injected

with LPS (500 pg/rnl) or carrier (PBS) ...................................................................... 59

List of Figures

Figure 1.1. Schematic representation o f the regulation of testicular steroid biosynthesis in

Leydig cells by endocrine, paracrine, and autocrine factors in fish ....................... . .... 10

Figure 2.1. Effect of graded doses of TNFa (1 - 100 @ml) on basal testosrerone of testis

exhibiting a GSI of A) 1.99 i 0.003 and B) 5.11 k 0.60. Testis pieces were incubated

for 18 hrat 18°C ......................................................................................................... 24

Figure 2.2. E ffect of graded doses of RIFa (O. 1 - 100 ng/ml) on hCG (0.2 IU1ml)-stimulated

testosterone production from testis pieces incubated for 18 hr at I8 O C .................. 25

Figure 2.3. Effect of graded doses of IL- 1 P (O. 1 - 1 O n g h l ) on basal testosterone production

from testis pieces incubated for 18 hrat 18°C .......................................................... 26

Figure 2.4. Effect of graded doses of IL- 1 P (0.0 I - 10 ng/ml) on hCG (0.2 IUlm1)-stimulated

testosterone production from testis pieces incubated for 1 8 hr at 1 8 " C ....... ....... . .... 27

Figure 2.5. Effect of TNFa (1 @ml) and IL4 P (1 ng/ml), alone and in combination, on

hCG (0.2 IU1ml)-stimulated testosterone production from testis pieces incubated for

18 hrat 18°C .............................,...............................................................+.............. 28

Figure 2.6. Effect of increasing concentrations of RTS l 1 macrophage conditioned media

(2.5-25% v/v) on basal testosterone production of testis pieces incubated for 18 hr at

........................................................................................................................ 1 8 T 29

Figure 2.7. Effect of increasing concentrations of RTS 1 1 macrophage conditioned media

(2.5-25% v/v) on hCG (0.2 IU/ml)-stimulated testosterone production from testis

.......................................................................... pieces incubated for 18 hr at 18°C 30

Figure 2.8. Immunoblocking ofTNFa inhibition of hCG (0.4 IUlm1)-stimulated testosterone

production by a mammalian TNFa antibody .......................................................... 3 1

Figure 2.9. Testosterone production by testis pieces stimulated with hCG (0.5 IU/ml) for 18

hr at 18 O C in the presence of absence of serum-free RTS I 1 supernatants (500 pi) that

were preincubated with antibodies to mammalian cytokines (1 0 pg, rabbit anti-mouse

TNFa and anti-human IL-lp), non-immunized rabbit serum (10 pg), or control

media ..................................................................................................................... ..3 2

Figure 2.10. Effect of TNFa (10 ng/ml) on A) basal and B) hCG (0.2 IU/ml)-stimulated

testosterone production by goldfish testis pieces incubated at 18°C for 2, 8, and

18hr 33

Figure 2.1 1. Effect of TNFa (10 nglml) on A) basal and B) hCG (0.2 IU/ml)-stimulated

testosterone production by goldfish testis pieces incubated at 18 OC for 2,8, and 1 8

Figure 2.12. Effects of RIFa on basal and hCG ( 1 UI/mI)-stimulated A) extracellular

cAMP and B) testosterone production by testis pieces incubated with I mM IBMX

for 1 and3 hrat 18°C .............................................................................................. 35

Figure 2.13. Effect of TNFa on vanous activators of steroidogenesis. Testis pieces were

incubated for 18 hr at 18 OC with hCG (0.2 IUfmI), forskolin (0.5 PM), and 8-bromo-

CAMP (0.15 mM) with and without TNFa (longiml) ........................................... 36

Figure 2.14. Effect of TNFa on the conversion of steroid precursors to testosterone. Testis

pieces were incubated with 25-hydroxycholesterol ( 1 and 10 yg/ml), pregnenolone

(50 and 100 nghl) , and 17a-hydroxyprogesterone (50 ng/ml) with and without

TNFa ( 1 O ng/ml). ................................................................................................... -3 7

Figure 2.15. Effect ofTNFa on testosterone production by testis pieces stimulated both with

............................... hCG (0.2 IU/rnl) and 25-hydroxycholesterol ( i and 10 pg/rnl) 38

Figure 3.1. Effect of graded doses of LPS (0.08- 10 @ml) on basal testosterone production

....................................................... fiom testis pieces incubated for 18 hr at 18 OC 57

Figure 3.2. Effect of A) low (0.2 pg/ml) and high (20 pg/ml) doses and B) 2 p g h l of LPS

on hCG (0.2 IU/ml)-stimulated testosterone production from testis picces incubated

...... for 18 hr at 18°C .............................................................................................. 58

Figure 3.3. Plasma testosterone levels in carrier (PM)- and LPS (500 pg/ml)-injected male

....................................................... çoIdfish with A) a GSI<1.0 and B) a GSIH.0 60

Figure 3.4. SDS-PAGE and Westem blot analyses with rabbit anti-mouse TNFa of proteins

in concentrated RTS 1 1 supernatants generated from 48 hr ce11 cultures with and

without lipopolysaccharide (20 pg/rnl) ................................................................... 6 1

Figure 3.5. SDS-PAGE and Westem blot analysis with rabbit anti-human IL- 1 f3 of proteins

in concentrated RTS 11 supernatants generated fiom 48 hr ce11 cultures with and

without lipopolysaccharide (20 pg/ml) .................................................................. 62

Figure 3.6. SDS-PAGE and Western blot analysis of A) anti-mouse TNFa antibody and B)

anti-human I L 4 b antibody of testis and kidney homogenates of fish used in Fig.

3.3 ............................................................................................................................ 63

Figure 3.7. Representation of SDS-PAGE and Western blot analysis of proteins from testis

and kidney hornogenates fiom control and LPS-injected goldfish ......................... 64

vii

CHAPTER 1

General Introduction

The regulation of testicular hnction in teleosts, as in mammals, is a cornplex

process involving not only the pituitary gonadotropins, but also a multitude of interacting

factors which act in an endocrine, paracrine, and autocrine marner (Van Der Kraak et al.,

1998). The involvement of immune-derived components, or immune-endocrine

interactions, on reproductive processes in fish is virtually unexplored. There are no

reports of the effects of cytokines on testicular steroid biosynthesis in teleosts, which is

not surprising given the current stanis of fish cytokine knowledge. Nonetheless, the

studies describing the effects of cytokines on testicular functioning in mammals, coupled

wi th the growing realization that immune-endocrine signalling is not restricied to higher

vertcbrates, lays the foundation for the hypothesis that cytokines are involved in the

regulation of testicular steroidogenesis in teleosts.

The purpose of this chapter is to provide an O-.,, icw d iiis synthesis and

regulation of androgen biosynthesis in teleosts and to discuss the potential involvement of

cytokines, namely IL- 1 and M a , in the regulation of testicular steroidogenesis in

vertebrates. The current knowledge of fish cytokines is included with a brief discussion

of their known invohement in teleost irnrnune-endocrine communication. The final

section describes thesis objectives and organization.

Testicular Androgen Biosynthesis and Regulation in Teleosts

In teleosts, Leydig cells are the predorninant sites of androgen synthesis in the

testis, as in mammals (Loir, 1990). The major androgens produced by the testicular

tissue Vary depending on developmental stage and species but may include testosterone

(T), 1 1-ketotestosterone (1 1-KT), and androstenedione (Fostier et al., 1987; Loir, 1990).

Salrnonids (e.g. rainbow trout) produce large amounts of 1 1 -KT (Shultz and Blum, 1 WO),

while goldfish have been shown to produce comparable arnounts of T and 1 1-KT (Wade

and Van Der Kraak, 1991). Androgens are involved in the regulatior. of a variety of male

reproductive functions including secondary sexual characteristics, gonadai differentiation,

spematogenesis. spenniation, and behaviour (spawning and parental care) (reviewed in

Fostier et al., 1987 and Redding and Patino, 1993).

Testosterone is synthesized fiom cholesterol through a variety of enzymatic steps

that occur in both the mitochondria and the smooth endoplasmic reticutum in Leydig cells

(refer to Fig. 1). The transport and mobilization of cholesterol into the inner

rnitochondrial membrane is a CAMP-dependent process involving the action of the

steroidogenic acute regulatory (StAR) protein. Although several other proteins may also

contribute (rcviewed in Stocco, 1996), StAR is believed to play a pivotal role in the acute

regulation of steroidogenesis in mammals (Thomson, 1998). The activities of teleost

StAR (55% amino acid homology to marnmals) are not as well described, but StAR has

been found to be expressed in trout steroidogenic tissues, inciuding the testis (Todo et al.,

2000).

Pnmarily, the endocrine control of testicular androgen biosynthesis is via the

pituitary gonadotropins, GtH I and II (see Fig. 1). These hormones are homologous to the

mammalian follicle stimulating hormone (FSH) and luteinizing hormone (LH),

respectively. Gonadotropin-releasing hormone (GnRH) secreted fkom the hypothalamus

2

stimulates the release of the gonadotropins which act upon the testes through specific

receptors on the surface of Leydig cells. Gonadotropin-stimulated testosterone

production occurs via the CAMP (protein kinase A) second messenger pathway, but may

also involve other intracellular signalling pathways such as phophatidylinositol

metabolites (inositol- 1.4,s-triphosphate and 1,2-diacylglceroi) and calcium (reviewed in

Van Der Kraak and Wade, 1994; Van Der Kraak et al., 1998).

The local, or paracrine, regulation of testicular steroidogenesis has been widely

studied in rnarnmals (Saez, 1994; Gnessi et al., 1997; Hales, 2000), but relatively lew

studies have examined the roles of intratesticular factors on reproductive processes in

teieosts. Contributing to this lack of knowledge, is the greater emphasis that is placed on

understanding the control of ovanan processes in this class. The growth factors,

epiderrnal growth factor a and transforming growth factor a, as well as steroids (e.g.

17B-estradiol) have been s h o w in v i m to inhibit gonadotropin-stimulated ovarian

steroid production in teleosts (Van Der Kraak et al., 1998). Arachidonic acid and its

metabolites, E series prostaglandins, stimulate ovanan and testicular T production in

goldfish (Wade and Van Der Kraak, 1993; Wade and Van Der Kraak, 1994; Van Der

Kraak et al., 1998) and are believed to be paracrine/autocrine regulators of gonadal

functioning. However, the relevance of locally produced cytokines to teleost gonadal

steroidogenesis and gametogenesis is virtually unknown. This is surprising given the

described activities of these signalling molecules in mammalian testicular (Hales, 2000)

and ovarian (Terranova and Rice, 1997) processes.

Cytokiîz es and Testicular Function in Mam mals: An Immune-Endocrin e Interaction

Communication between the immune and endocrine systems in vertebrates is

facilitated, in part, by the activities of cytokines (Balm, 1997; Saez, 1994). The

proinflammatory cytokines, IL-1 and TNFa, act as multifunctional messengers in both

systems. These cytokines play a central role in stimulating immune and infiammatory

processes in response to microbial infection and tissue injury (Rock and Lowry, 199 1;

Dinarello, 1988). In addition to their well characterized roles within the immune system,

TNFu and IL- 1 have been shown to regulate testicular function in marnmals. Testicular

function is altered during conditions of inflammation and infection (e.g. sepsis,

rheumatoid arthritis. bum trauma) (Handelsman, 1994; Martens et al., 1994) and it is

generally believed that the induction of cytokines contributes to the alteration (revicwed

in Hales, 2000). However, the regulation of testicular steroidogenesis and

spennatogenesis dunng normal physiological conditions by cytokines, when they may act

as growth and differentiation factors (Khan et al., 1992a; b), is more debatable (Moore

and Hutson, 1994; Cohen and Pollard, 1998).

The mature, secreted mammalian TNFa (approximately 17 kDa) peptide belongs

to the TNF ligand superfamily and exerts its activity through either of two distinct cell

surface receptors: TNF-RI and RIF-RI1 (Idriss and Naismith, 2000). IL-1 is a family of

three distinct 17 kDa peptides: IL4 a, IL- 1 P, and IL- 1 receptor antagonist (IL- 1 Ra).

There are two p n m q ce11 surface receptors for IL-1, however, only one receptor (IL-

1 RI) transduces a signal while the other (IL-I NI) is a decoy receptor (Dinarello, 1997).

IL- 1 a and IL- 1 P overlap considerably in their biological activities which are often similar

to the activities of m a . While their predominant source is from activated monocytes

and macrophages, a variety of ce11 types throughout the body are known to synthesize IL-

1 and TNFa including other immune (e.g. neutrophils) and non-immune cells (e.g.

endothelial) (Aggarwal and Reddy, 1994; Dinarello, 1988). The predominant

intratesticular source of TNFa and IL- 1 is activated macrophages (Xiong and Hales,

1993a; Hutson, 1994; Kem et al., 1995), although studies have indicated that germ cells

(De et al., 19931, Sertoli cells (Gerard et al., 1992), and Leydig cells (Gnessi et al., 1997)

are potential sources of cytokines. In males, the intimate association of Leydig cells and

resident testicular macrophages in the interstitiai tissue of the testis suggests that an

important functional relationship exists between these cells and their secreted products

(Hutson. 1994).

Lipopolysaccharides (LPS, bacterial endotoxin) are potent inducen of IL- 1 and

TNFa release. Endotoxins exert their effects indirectly through the induction of cellular

mediators (e.g. cytokines, oxygen fiee radicals, prostaglandins) and can have eithrr

beneficial or harmfûl effects on the host organism (Rietschel and Brade, 1992). LPS

administration, in vivo and in vitro, results in the secretion of IL4 and TNFa from

testicular intentitial macrophages in mice (Hales et al., 1992; Xiong and Hales, 1993b;

Xiong and Hales, 1994). Several studies have observed decreased T levels, decreased

levels of pituitary gonadotropins, and dismption of spermatogenesis in animals treated

with LPS (Wallgren et al., 1993; O'Bryan et al., 2000a; Hales et al., 2000). The

inhibition of Leydig ce11 steroidogenesis during experimental endotoxemia has been

found to occur ptimatily at the level of the testes (O'Bryan et al., 2000a, Hales et al.,

2000) and involves the supression of the steroidogenic enzymes cholesterol side-chain

cleavage cytochrome P450 (P450scc). 3P-hydroxysteroid dehydrogenase (3P-HSD), and

17a-hydroxylaseK 17,20-lyase cytochrome P450 (P450c 17) (Xiong and Hales, 1994;

Bosmann et al., 1996), which rnay occur concomitant with a decline in StAR production

(Bosmann et al., 1996). There is strong evidence to suggest that cytokines are key

mediators in the activities of LPS, at least in cases of long-term steroid inhibition

(reviewed in Hales, 2000).

Numerous studies have examined the effects of TNFa and IL- 1, as well as

macrophage-conditioned media on testicular testosterone production (Saez, 1994; Gnessi

et al., 1997; Hales, 2000) in mammals. Studies by Sun et al. ( 1993) and Sun and

Risbridger ( 1994) found that rat testicular macrophage conditioned media (TMCM)

inhibited LH-stimulated T production but had no effect on basal levels. However, these

results are in contrast to a previous report by Yee and Hutson (1985) that demonstrated a

stimulatory effect of rat TMCM on both basal and LH-stimulated Leydig cells. Similarly,

most reports have found that IL4 and TNFa inhibit gonadotropin (hCG, LH) and CAMP-

stimulated T production in a variety of Leydig ce11 cultures, including porcine (Mauduit

et al., 199 l), mice (Xiong and Hales, 1993b; Xiong and Hales, 1994), and rat (Lin et al.,

1994; Li et al., 1995). The cytokines affect basal T production more variably with reports

ranging from no effect to stimulation or inhibition of T (Hales, 1996; Xiong and Hales,

1998).

The pleiotrophic nature of IL- 1 and TNFa is demonstrated well by their multiple

sites of action in the steroidogenic pathways. Although the steroidogenic enzymes

(P45Oc 17. P450scc, and 3P-HSD) appear most sensitive to these cytokines, TNFa has

also been reported to act proxirnally to CAMP activity through the inhibition of

gonadotropin binding and CAMP formation (Mauduit et al., 199 1). The delivery of

choIestero1 to the inner mitochondrial membrane may also be disrupted by TNFa, but not

IL- 1 a (Lin et al., 1998), as TNFa has been found to decrease both StAR gene expression

and protein çythesis (Mauduit et al., 1998; Budnik et al., 1999) in rodent Leydig cells.

Teleost Cytokines and Immune-Endocrine Interactions

In the past decade, several approaches have been taken to investigate cytokine

activity in fish (reviewed in Secombes, 1996). These research efforts are dnven

predominantly by the prospects of improving disease resistance through cytokine-

mediated modulation of immune responses (Secombes et al., 1999a;b) and have not

focussed on their possible roles in reproductive processes. Recently the gene sequences

for IL- 1 B from several teleosts, including Salmon, rainbow trout (Secombes et al.. 1998;

Zou et al., 1999a) and carp (Fujiki et al.. 2000) have been identified. Rainbow trout IL-

1 p shows between 49 and 56% amino acid similarity to mammalian IL- 1 B (Zou et al.,

1999b) and gene transcripts have been detected in a variety of tissues (ie. blood, gill,

liver, kidney, and spleen). It is not known whether IL4 f3 is expressed and synthesized in

gonadal tissues of fish. Also, the gene for TNF in Japanese flounder (Hirono et al., 2000)

and brook trout (Bobe and Goetz; 2001) has been sequenced. Although the flounder TNF

amino acid sequence is 29% and 3 1% similar to human TNFa and human lymphotoxin a,

respectively, Hirono et al. (2000) believe the teleost TNF gene more closely resembles

mammalian TNFa. Interestingly, Bobe and Goetz (in press) f ond TNF to be expressed

in ovanan and testicular tissues, while Hirono et al. (2000) did not. Further work is

required to discern whether or not these cytokines are expressed in teleost gonadal

tissues.

There is convincing evidence for bidirectional communication between the

immune and endocrine systems in teleosts. This includes the observation that immune

activation (i.e. IL- 1 P, LPS) of the hypothalamus-pituitary-interrenal (H-P-1) mis leads to

elevated glucocorticoid secretion ( B a h , 1997; Holland et al., 2000). As well, hormones

(e.g. sex steroids, growth hormone, cortisol) modulate responses of immune cells ( B a h ,

1997; Weyts et al., 1999). While support for the idea of an interactive cytokine network

rrpulating immune and endocrine responses in fish is growing, there are no snidies that

have investigated the effects of proinflamrnatory cytokines on testicular (or ovarian)

functioning in teleosts. The potential for the paracrine regulation of testicular

steroidogenesis by cytokines in teleosts is considerable. Not only is there great homology

in the pathways (and endocrine regulation) of steroidogenesis between vertebrate classes,

the basic organization of the testis is similar, as well. Macrophages and Leydig cells are

present in the interstitial tissue of the testis in teleosts (Loir et al., 1995), thereby allowing

the hypothesis to be formed that cytokines secreted fiom macrophages interact with

neighbouring cells to modulate steroidogenesis and testicular functioning.

Thesis Organization and Objectives

The first objective of my study (Chapter 2) was to investigate the effects of the

proinflammatory cytokines, TNFa and IL- 1 P, and macrophage-denved products on T

production by goldfish testis pieces. As responses to the cytokines were easily obtained,

the work involving TNFa was extended to include an investigation into its possible

mechanism(s) of action. This was assessed by evaluating the CAMP signal transduction

pathway, as well as stimulating T production by known activators of steroidogenesis to

discrm the site(s) of action within the pathway. In the absence of available recombinant

fish I L 1 P and TNF, murine recombinant IL-1 P and TNFa were used. The rainbow trout

spleen macrophage ce11 line, RTS 11. provided a potential source of teleost cytokines.

The second objective (Chapter 3) was divided into 2 parts. The first part

evaluated the effects of LPS on T levels in vivo and in vitro in the male goldfish.

Secondly. the production of IL-! and TNF-like peptides by teleost macrophages (RTS 1 1

cells) and testicular tissue were investigated by immunodetection using antibodies to

mammalian IL- 1 P and TNFa. These objectives provide an evaluation of the potential

regulation of testicular steroidogenesis by immune-derived molecules in teleosts which

contributes to the overall understanding of immune-endocrine interactions in fish.

Fig. 1 . Schematic representation of the regulation of testicular steroid biosynthesis in

Leydiç cells by endocrine, paracnne, and autocrine factors in fish. Testosterone (T) is

formed from cholesterol which undergoes a senes of enzymatic steps. The enzymes

essential for the production of T are cholesterol side-chain cleavage cytochromc P450

(P4jOscc). 3P-hydroxysteroid dehydrogenase (3P-HSD), 17a-hydroxylasdc 17,204yase

cytoc hrorne P450 (P450c 1 7), and 1 7P-hydroxysteroid dehydrogenase ( L 7P-HSD).

Endocrine Control

T Paracrine Control

3 p h m hhydrwpi- 17a-Hydroxy- androsterone progesterone

I

Testosterone

CHAPTER 2

III Vitro Modulation of Goldfish Testicular Testosterone Production by

Tumor Necrosis Factor a, Interleukin-lp, and Macrophage-Derived Products

INTRODUCTION

Reproductive processes are under the complex and interacting control of both the

immune and endocrine systems. It is now recognized that testicular steroid biosynthesis

is not only under pituitary gonadotropin regulation, but also under local control by

cytokines. traditionally described as immunoregulatory peptides. These polypeptides are

a broadly defined group of intercellular mediaton most studied for their fùnctional roles

as chernical signals between vanous cells of the immune system (Ben-Rafael and

Orvieto, 1992). The proinflammatory cytokines, tumor necrosis factor a (TNFa) and

interleukin- 1 (IL- I), have been impiicated in the regulation of mamrnalian testicular

Functioning under pathophysiological and normal conditions (Martens et al., 1994;

Hutson, 1994). Elevated levels of proinflarnmatory cytokines as a result of systemic

infections (e.g. sepsis) or inflarnmatory diseases (e.g. rheumatoid arthritis) occur

concomitantly with decreased serum testosterone levels. Leydig ce11 growth and

di fferentiation during normal development has been shown to be stimulated by IL- 1 and

TNFa and in modeis of testicular macrophage depletion, the development and

functioning of Leydig cells is impaired (reviewed in Hales, 2000).

The mature, secreted TNFa (approximatelylï D a ) peptide belongs to the TNF

ligand superfamily and exerts its activity through either of two distinct cet1 surface

receptors: TNF-RI and TNF-NI (Idriss and Naismith, 2000). IL- 1 exists as two foms,

IL- 1 a and IL- 1 P (both approximately 17 kDa), that overlap considerably in their

biological ac tivities (Dinarello, 1997). Principally, the intratesticular source of TNFa

and IL- I is activated resident macrophages (Xiong and Hales, 1993; Hutson, 1994; Kem

et al., 1995), although studies have indicated that germ cells (De et al.. 1993), Sertoli cells

(Gerard et al., 1992), and Leydig cells (Gnessi et al., 1997) are potential sources of

cytokines. In males, the intimate association of Leydig cells and resident testicular

macrophages in the interstitial tissue of the testis suggests that an important functional

relationship exists between these cells and their secreted products (Hutson, 1994). There

is ovenvhelming evidence in rnarnmals for the involvement of cytokines in male gonadal

functioning (Saez, 1994; Gnessi et al., 1997; Hales, 2000) and numerous studies have

cxamined the effects of TNFa and IL- 1, as well as MCM on testicular testosterone

production (reviewed in Hales, 1996 and Hales, 2000). Although a few inconsistencies

exist in the literature, there is a general consensus that proinflammatory cytokines inhibit

gonadotropin or CAMP-stimulated steroidogenesis in mammalian Leydig cells, while

basal steroid production is more variably affected.

The current knowledge of cytokines in teleosts lags far behind marnmalian studies

and therefore, it is not surprising that the roles of fish cytokines in mediating immune-

endocrine interactions are relatively unexplored. Work by Loir et al. (1995) on rainbow

trout determined that testicular macrophages are nurnerous in the interstitial tissue and

tubules of regressed or resuming sperrnatogenesis testes, but that very few macrophages

are present in spermatogenic and spermiating testes. Similar to mammals, Leydig cells

reside in the interstitial tissue (Grier, 198 1) and are the predominant source of androgens

in teleosts (Loir, 1990). Therefore, the potential exists for cell-ce11 interaction whereby

cytokines secreted by macrophages may exert an effect on neighbounng testicular cells.

This hypothesis is supported in one report by Loir et al. (1995) that discerned that factors

secreted by trout testicular macrophages were both stimulatory or inhibitory on DNA

synthesis in spermatogonia and spermatocytes depending on the macrophage preparation.

Although thrre are no reports examining the effects of cytokines on gonadal steroid

biosynthesis in fish, immune-activation via LPS and injection of both recombinant

rnammalian and trout IL- I P has been shown to affect vanous physiological parameters,

including activation of the interrenal axis in teleosts (White and Fletcher, 1985; Balm et

al., 1995; Balm, 1997; Weyts et al., 1999; Holland et al., 2000). In the absence of

availabie fish recombinant cytokines, several studies have shown that mammalian TNFa

and IL- 1 can cross-react with fish leukocyes (Hardie et al., 1994; Jang et al., 1995a:

Secornbes et al., 1996) and the antigenic cross-reactivity of mammalian anti-lL- 1

antibodies has been dernonstrated in Western blots of fish leukocyte supernatants

(Ellsaesser and Clem, 1994; Verburg-van Kemenade et al., 1995). Recently, the genes

for Japanese flounder TNF (Hirono et al., 2000) and trout (Zou et al., 1999b) and carp

(Fujiki et al., 2000) IL4 P were sequenced.

The objectives of the present study were: (1) to investigate the ability of

mammalian TNFa and IL- I P and fish macrophage-denved products to modulate basal

and gonadotropin-stimuiated testicular testosterone (T) production by goldfish testis

pieces in vitro, (2) to determine if the activity of the macrophage-denved factor(s) could

be nullified by antibodies against mammalian TNFa and IL- 1 P, and (3) to evaluate the

site(s) in the steroid biosynthetic pathway where TNFa affects gonadotropin-induced

testosterone production.

MATERIALS AND METHODS

Fish

Common goldfish were purchased from DAP International (Etobicoke, ON) and

were maintained at the Hagen Aqualab, University of Guelph, in 1.8 m diameter circular

tanks with fiow through water at 16-1 8 O C under a constant photoperiod (14 hr light/ 10 hr

dark). Fish were fed once daily to satiation.

Cltent icals, Hormones and Antibodies

Fonkolin, 25-hydroxyc holester01 (250H-chol), 8-bromo-CAMP ( 8-br-CAMP),

pregnenolone, 17a-hydroxyprogesterone ( 17aOH-progesterone), human chononic

gonadotropin (hCG), and 3-isobutyi- 1 methylxanthine (IBMX) were purchased from

Sigma Chernical Co. (St. Louis, MO). Leibovitz L-15 media with L-glutamine.

penicillin, streptomycin, and fetal bovine serum were purchased from Gibco BRL

(Burlington, ON). Murine recombinant tumor necrosis factor a (TNFa) and interleukin-

1 p (IL- 1 P), and affinity purified polyclonal rabbit anti-mouse TNFa antibody were

purchased from Chemicon International, Inc. (Temecula, CA). The polyclonal rabbit

anti-human IL-1 B antibody was bought from Upstate Biotechnology (Lake Piacid, NY).

IBMX and hCG were dissolved directly into L- 15 media while the steroids were first

dissolved in 95% ethanol and subsequently diluted with L-15 prior to use. The volume of

ethanol in experimental and control groups did not exceed 1% of the final incubation

volume which has been shown to not affect basal or stimulated testosterone levels (Van

Der Kraak and Chang, 1990). Fonkolin was initially dissolved in DMSO and then

diluted with L- 15 before use in the incubations. The final concentration of DMSO in the

incubations did not exceed 0.5% which has been shdwn to not affect basal or stimulated-

testosterone production (Wade and Van Der Kraak, 199 1). Cytokines and antibodies

were reconstituted in sterile distilled water and subsequently diluted in L- 15 prior to their

addition to the testis incubations.

Testis Incubaiiorts

Goldfish testis incubation procedure was modified from Wade and Van Der Knak

( 199 1). Briefiy, male goldfish were killed by spinal transection and the testes were

placed in L- 15 media supplernented with penicillin (200 U/ml) and streptomycin (200

m l ) . In most experiments, the gonadosomatic index (GSI; GSI = gonad weightl (body

weight-gonad weight) X 100) range of the goldfish used was 3-6% (average 4.6% * 0.2).

This GSI range is indicative of testes in an advanced stage of the spematogenetic cycle

in cypnnids (Billard et al., 1982). A larger GSI range was used in four experiments

conducted to determine the effects of TNFa on basal T levets, The connective tissue was

removed and two testis pieces of approximately equal size weighing a total of 18-24 mg

were placed in borosilicate tubes (12 X 75 mm). Individual expenments were conducted

with tissues from one or two fish, depending on the arnount of tissues required. In

experiments using tissues from 2 goldfish, each tube contained one piece fiom each fish.

The media was replaced with fresh L-15 prier to the addition of test compounds.

Typically, there were 3-4 replicate incubations per treatment in a 1 ml final volume.

AR er the incubation, the tubes were centnfuged for 5 min at 2000g and the medium was

decanted and stored at -20°C p io r to the measurement of T levels by radioimmunoassay

(RIA). T measurement by RIA was previously described by Van Der Kraak and Chang

( 1990).

cAMP A tialyses

Similar procedures to Wade and Van Der Kraak (1993) were used in experiments

cxamining the production of cAMP released to the incubation media. In these

incubations, the tissue was pre-incubated for 2 hr in L- 15 media at 18 OC to allow cAMP

levels to stabilize. Prior to the addition of test compounds, fresh L- 15 containing 1 mM

IBMX was added to the tissue to prevent the degradation of endogenous CAMP by

phosphodiesterases. AAer incubation, tubes were centrifiged for 5 min at 2000rpm and

half of the media was boiled for 10 min and stored at -20°C unril analysis of CAMP by

RIA (CAMP Kit, Biomedical Technologies Inc., Stoughton, MA). The remaining

incubation media was fiozen at -20°C for later T analysis.

Preparation of R T ' l l (trout spleen macrophage ceil iine) Supernatan b

RTS 1 1 cells (Ganassin and Bols, 1998) were maintained in continuous culture at

the University of Guelph in L- 15 medium supplemented with 25% fetal bovine semm and

penicillin/streptomycin ai 18°C without supplemental COZ Prior to use, cells were

harvested from tissue culture fiasks by gentle scraping and centrifùged at 1000 rpm for 5

min. The supernatant was aspirated and replaced with serum-free L- 15 media. Cells

were then cultured at 500 000 cells/ml in 24 well plates for 24 hr at 18°C. The MCM

was coilected, spun for 5 min at 200g to pellet any debris, and fiozen at -20°C pnor to

use. Typically, the fiozen samples were used in the incubations within 4 weeks. The

17

activity of the supematants on T production in the incubations was tested over the range

of O-25% V/V.

Antibody-lnliibilion Studies

In the control experiment, 50 ng of TNFa was preincubated with 20 pg of

polyclonal rabbit anti-rnouse TNFa antibody, 20 pg of non-immune rabbit serum, or

control media for 3 hr at 18°C in a final volume of LOO pl. After the preincubation, 20 pl

of the preincubated mixtures were added to the incubation tubes in conjunction with hCG.

The MCM experiment was conducted similarly, except that 500 pl of MCM was pre-

incubated with 10 pg of either rabbit anti-mouse TNFu and IL- 1 P, 10 pg of non-immune

rabbit serum, or control media. The mixtures (100 pl) were added to the incubation

media in conjunction with K G .

Sfatistical Analyses and Presentation of Data

Most experiments are represented as standardized data to allow combining of

repeated experiments for graphing and statistical purposes. T levels were convened to %

of basal or ?4 of stimulated T production. The differences between treatment means of

combined data were compared using General Linear Model procedures (proc GLM; SAS

Institute Inc., Cary, NC) and when warranted, followed by Tukey's HSD post hoc test. A

p value of <O.OS was considered significant. T levels that were below the detection limit

of the RIA were assigned the detection limit value of 15.6 pg/ml for statistical purposes.

When more than one repiicate was non-detectable in a treatment, the entire treatment was

deemed non-detectable.

RESULTS

Effects of TNFa; IL-laand macrophage- derivedproducts on basal and K G - stimulated testosterone production

In the absence of the availability of fish cytokines, the initial experiments

examined the effects of murine recombinant tumor necrosis factor-a (RIFa) and murine

recombinant interleukin- 1 P (IL- 1 P) on basal and hCG-stimulated T production. Basal T

levels were differentiaily affected by TNFa in experiments that used testis that varied

considerably in GSI values (Fig. 2.1). With GSI as the covariant in an ANCOVA, the

GSI effect is significant (p=0.0001) and so the data were divided into two groups, fish

with low GSI (1.99 k 0.003) and fish with high GSI (5.14 * 0.60). In fish with low GSI,

TNFa potentiated basal T levels (p=0.0001), but only at the lowest concentration tested

( 1 ng/ml). Whereas in fish with high GSI, TNFa (1-100 ng/ml) inhibited T (p=0.0006).

The diametric response of the tissue to TNFa is suggestive of a cyclical effect that may

be dependent upon the reproductive stage of the testis. Figure 2.2 demonstrates the

inhibitory effect of TNFa (O. 1- 100 @ml) on hCG (0.2 IU/ml)-stimulated T (p=0.000 1)

with a maximal inhibition of 32%.

Basal T (Fig. 2.3) was inhibited by IL- 1 P over the range of O. 1-10 @ml in a non-

dose dependent manner (p= 0.0004). Likewise, Fig. 2.4 (n= 4) indicates that IL- 1 P (0.0 1 -

10 ng/ml) caused a consistent and significant inhibition of hCG (0.2 IU/ml)-stimulated T

production (p= 0.0001) with a maximal inhibition of 36%. There was no significant

additive effect on the inhibition of hCG (0.2 IU/ml)-stirnulated T when low doses of

TNFa (1 .O ng/ml) and IL-1 P (1 .O ndml) were used in conjunction (Fig. 2.5). Although, 3

out of 4 experiments showed that the combined treatment group (TNFa + IL-1P) resulted

in greater inhibition than either cytokine alone. The combined treatment resulted in a

3 1 % inhibition of gonadotropin-stimulated T production, while TNFa and IL- 1 P alone

caused a 20% and 14% decrease, respectively.

To compare the effects of mammalian cytokines with fish macrophage-derived

products on T production, goldfish testis pieces were incubated with varying amounts of

RTS 1 1 culture supematants (macrophage-conditioned media; MCM). The results of the

srudy examining the effects of the MCM (2.5-25% v/v) on basal T production are shown

in Figure 2.6. The MCM had no effect on basal T production. Conversely, Figure 2.7

shows that the lower doses of MCM (2.5-5% v/v) inhibited hCG (0.2 IU/ml)-stirnulated T

in a biphasic manner (p= 0.0003). Similar to the effects of the mammalian cytokines, the

maximal inhibition was not drastic (36%).

E m s of un tibodies to TNFaand ILIPon MCM activity

The similar responses observed between the cytokines and the MCM, coupled

with the fact that macrophages are the predominant source of TNFa and IL- 1 P in

mammals (Dinarello, 1988; Rock and Lowry, 199 l), led to an investigation to determine

if the inhibitory activity found in RTS 11 supematants could be due to the presence of

TNF- and/or IL 1-like molecules. Initially, one control experiment (Fig. 2.8) was

conducted with a cornrnercially available rabbit anti- mouse M a antibody to ensure

that the activity of M a could be nullified in this goldfish testis system. The hCG (0.4

IUim1)-stirnulated T production was inhibited by TNFa, and indeed, the antibody

nulli fied this activity (p= 0.0034). The control, non-immunized rabbit senun (NIRS), did

not interfere with the level of hCG stimulation and did not affect the TNFa inhibition of

hCG-stimulated T. Commercially available rabbit anti-mouse WFa and anti-human IL-

1 p antibodies were preincubated with RTS I 1 supernatant in an attempt to nulliQ the

activity of the MCM. As illustrated in Figure 2.9, both TNFa and IL- 1 B antibodies

inhibited hCG (0.5 IU/ml)-stimulated T production (p=O.OO 13). This effect precluded

drawing conclusions as to whether or not the active factors in the MCM were

antigenically similar to murine TNFa or human IL- 1 B. Antibody-inhibition experiments

using anti-TNFa were conducted another 4 times with slight methodical variations and

each experiment was unsuccessful in nuHiQing the MCM activity.

T N F a siteCs) of action

Several experiments were perforrned to determine the mechanism by which TNFa

inhibits T production in the goldfish testes incubations. A time course experiment (Fig.

2.10) indicatcd that the decline in (A) basal and (8) hCG (0.2 IU/ml)-stimulated T levels

is greatest at 18 hr afler M a (10 ng/ml) treatment. At 18 hr, basal T was inhibited by

66% (p=0.0009) and KG-stimulated T was moderately decreased by 25% (p=O.O 135).

The repeat of this expenment is s h o w in Fig. 2.1 1. In this case, TNFa moderately

inhibited hCG-stimulated T at the 2 , 8 and 18 hour time points (p=0.0387, p=0.0 147,

p=0.0328, respectively). Maximal inhibition of hCG-stimulated T production was

achieved at 18 hr (36%). TNFa had little effect on basal levels throughout the time

course. however, there was a significant decrease in T levels at 8 hr (p=0.02 12).

In two separate experirnents (representation Fig. 2.12), basal and gonadotropin-

stimulated (A) extracellular CAMP and (B) T production were measured in the presence

and absence of TNFa ( 1 00 @ml). As CAMP levels change rapidly in response to

gonadotropin stimulation, the levels of c W in the media was measured after 1 and 3

hours of incubation. To prevent the breakdom of CAMP by phosphodiesterases, al1

treatments contained I mM IBMX. As expected, the treatment of the testis with hCG

resulted in significant increases in both media cAMP and T production within the first

hour of incubation and these levels were further elevated at 3 hr compared to the controis.

Although TNFa did not affect basal or hCG-stimulated CAMP levels at either time

period. TNFa did result in a srna11 but significant inhibition of hCG-stimulated T

production by 3 hr.

The nonnalized data in the remaining experiments in this section were generated

from four different fish. The average level of T produced by untreated testis in these

experiments was 10 1.3 + 4.7 pglml (data not shown). Treatment groups are s h o w

together based on the similar propeities of the steroidogenic activators. Fig. 2.13

demonstrates that T production from testis pieces stimulated with hCG (0.2 IU/ml),

forskolin (0.5 mM) which activates adenylate cyclase, and a CAMP analog, I-bromo-

cAMP (0.1 5 mM) in the presence of TNFa ( 1 O @ml) was inhibited by 3 1 % (p=0.0006),

3 1% (p=0.0049) and 37% (p=0.0008), respectively. Combined, these data indicate that

TNFa affects androgen production distal to CAMP formation and activity.

Subsequently, experirnents were conducted to determine whether TNFa may

affect the conversion of sterol substrates to testosterone. It is shown in Fig. 2.14 that

TNFa had a small potentiating effect (15%) on 250H-chol(1 pg/ml, p=0.043), a

tendency to inhibit (17%) pregnenolone (100 ng/ml, p=0.02), and did not affect 17 a-OK-

progesterone (50 ng/ml) stimulated levels of T. Figure 2.15 shows that the addition of

250H-ch01 (a cholesterol substrate denvative which readily passes through ce11 and

rnitochondrial membranes) at 1 and 10 pg/ml was unable to prevent the inhibitory action

of the cytokine on the gonadotropin action. M a treatment resulted in a maximal

inhibition of 28% on the hCG with 25OH-chol(1 pg/ml)-stimulated levels of T.

Together, these data suggest that the major inhibitory effect of TNFa on the gonadotropin

action occurs at a step(s) related to cholesterol substrate availability in the mitochondna

rather than to the sterol conversion to androgens.

TNF a (ng/ ml)

Fig. 2.1. Effect of graded doses of tumor necrosis factor a (TNFa; 1-100 ng/rnl) on basal

testosterone (T) of testis exhibiting a gonadosomatic index value of A) 1.99 0.01 and B)

5.14 i 0.60. Testis pieces were incubated for 18 hr at 18 O C . Values represent the mean k

SEM of 2 fish each with 4 replicate incubations per treatment. The Ievel of T of the

conrrol group in (A) and (B) was 3 1.9 * 2.1 pgiml and 46.1 * 5.2 pg/ml, respectively.

Values designated by the same letter are not significantly different fiom each other

(Tukey's test, p< 0.05).

TNFa O ( n m o

hCG (0.2 IUIml)

Fig. 2.2. Effect of graded doses of tumor necrosis factor a (TNFa; O. 1 - 100 ng/ml) on

hCG (0.2 IU/ml)-stimulated testosterone (T) production from testis pieces incubated for

18 hr ai 18'C. Values represent the mean * SEM of 3 fish each with 3 replicate

incubations per treatment. The level of T in the control group was 20 17.0 k 12 1.3 pg/ml.

Values designated by the same letter are not significantly different fiom each other

(Tukey's test, p< 0.05).

IL-IP (ng/ ml)

Fig. 2.3. Effect of graded doses of interleukin- 1 P (IL- 1 P; 0.1 - 1 O ng/ml) on basal

testosterone (T) production from testis pieces incubated for 18 hr at 18 "C. Values

represent the rnean * SEM of 2 fish each with 4 replicate incubations per treatment. The

level of T in the control group was 46.1 * 5.2 pg/ml. Values designated by the same

letter are not significantly different fiom each other (Tukey's test, p< 0.05).

Fig. 2.4. Effect of graded doses of interleukin- 1 B (IL- 1 P; 0.0 1 - 1 O @ml) on hCG (0.2

IU/ml)-stimulated testosterone (T) production from testis pieces incubated for 18 hr at

18 O C . Values represent the mean * SEM of 4 fish each with 4 replicate incubations per

treatment. The level of T in the control group was 2443.8 k 23 1.2 pg/ml. Values

designated by the same letter are not significantly different fiorn each other (Tukey's test,

p< 0.05).

TNFa IL-Ip

+ + TNFa

+ IL-1 p +

Fig. 2.5. Effect of tumor necrosis factor a ( M a ; 1 @ml) and interleukin- 1 B (IL- 1 B; 1

ngiml), alone and in combination, on hCG (0.2 IU/ml)- stimulated testosterone (T)

production. Testis pieces were incubated for 18 hr at 18 O C . Values represent the mean +

SEM of 4 fish each with 4 replicate incubations per treaiment. The level of T in the

control group was 2 146.4 * 278.7 pg/ml. Values designated by the same letter are not

significantly different from each other (Tukey 's test, p< 0.05).

% Macrophage- Conditioned Media

Fig. 2.6. Effect of increasing concentrations of macrophage conditioned media (2.5- 25

% viv) from the RTS 1 1 ce11 line on basal testosterone (T) production. Testis pieces were

incubated for 18 hr at 18°C. Values represent the mean k SEM of 2 fish each with 4

replicate incubations per treatment. The level of T in the control group was 1 12.3 k 18.3

pg/ml. Values designated by the same letter are not significantly different from each

other (Tukey's test, p c 0.05).

Fig. 2.7. Effect of increasing concentrations of macrophage conditioned media (MCM;

2.5-25% viv) from the RTS 1 1 ce11 line on hCG (0.2 IU/ml)-stimulated testosterone (T)

production from testis pieces incubated for 18 hr at 18 O C . Values represent the mean I

SEM of 2 fish each with 4 replicate incubations per treûtrnent. The level of T in the

control was 2023.3 * 196.1 pg/ml. Values designated by the same letter are not

si pi ficantly different from each other (Tukey's test, p< 0.05).

a O-TNF + T N F

Controi Anti- TNFa NIRS

Fig. 2.8. Immunoblocking of tumor necrosis factor a (TNFa) inhibition of hCG (0.4

IU/ml)-stimulated testosterone (T) production by a TNFa antibody. All test compounds

were pre-incubated for 3 hr at 18'C before being incubated with testis pieces for 18 hr at

18 O C . TNFa (50 ng) was preincubated with rabbit anti-TNFa (20 pg), non-immune

rabbit semm (NIRS; 20 pg) or control media pnor to being added to the testis pieces with

hCG. Final concentrations of M a , a n t i - m a , and NIRS were 10 ng/ml, 4 pg/ml, and

4 @mi, respectively. Values represent the mean i SEM of one fish with 4 replicate

incubations per treatment. Values designated b y the sarne letter are not signi ficantly

different from each other (Tukey's test, p< 0.05).

a O - MCM

T I+ MCM

Con trol Anti- TNFa

Anti- Anti-TNFa NIRS L-IP + Anti-ILlB

Fig. 2.9. T estosterone (T) production by testis pieces stirnulated with hCG (0.5 IU/ml) for 18 hr at

18 "C in the presence or absence of serurn-free RTS 1 1 macrophage conditioned media (MCM, 500

pl) that were pre- incubated with antibodies to rnammalian cytokines (10 pg, rabbit anti- mouse

TNFa and anti-hurnan I L 4 fi), non-immunized rabbit semm ( 1 O pg, NIRS), or control media. The

final concentration of the MCM, the cytokine antibodies, and NlRS in the incubation, was 10%

(vlv), 2 pg/rnl, and 2 pg/ml, respectively. Values represent the mean * SEM of one fish with 4

replicaie incubations per treamient. Values designated the same letter are not significantly

different from each other (Tukey's test, 0.05).

h

E \ an e V

c! E a C W. C C El:

C

Fig. 2.10.

2000 1 + hCG + TNF

90

Incubation Time (Hours)

80 -

Effect of tumor necrosis factor a (TNFa; I O ndml) on (A) basal and (B) hCG

A -+- Control -

(0.2 IU/ml)-stimulated testosterone (T) production by goldfish testis pieces incubated at

1 S O C for 2, 8, and 18 hours. Levels below the detection limit are represented by "ND".

Values represent the mean k SEM of one fish with 4 replicate incubations per treatrnent.

Astetisks represent T levels that are significantly lower than non-TNFa treated levels at

the same time period (ANOVA, p< 0.05).

A + Basal

+ + TNF

*

B + hCG

-t hCG + TNF

Incubation Time (Hours)

Fig. 7.1 1 . Effect of tumor necrosis factor a (TNFa; 10 nglml) on (A) basal and (B) hCG

(0.2 IU/ml)-stirnulated testosterone (T) production by goldfish testis pieces incubated at

18 "C for 2, 8, and 18 houn. Values represent the mean k SEM of one fish with 4

replicate incubations per treatment. Asterisks represent T levels that are significantly

lower than non-TNFa treated levels at the same time period (ANOVA, p< 0.05).

1 3

Incubation Time (Hours)

Fig. 2.12. Effects of tumor necrosis factor a (TNFa) on basal and hCG ( 1 IU/ml)-

stimulated (A) extracellular CAMP and (£3) testosterone (T) production by testis pieces

incubated with 1 mM IBMX for 1 and 3 houn at 18°C. Values represent the mean 5

SEM of one experirnent with four incubations per treatment. Values designated by the

same letter are not significantly different from each other (Tukey's test, p< 0.05).

35

O Control

hCG Forskolin 8-btomo-CAMP

Fig. 2.1 3. Effect of tumor necrosis factor a ( M a ; 1 0 ng/ml) on various activators of

steroidogenesis. Testis pieces were incubated for 18 hr at 18 O C . The levels of

testosterone (T) afler treatment with hCG (0.2 IUIml), forskolin (0.5 PM), and 8-bromo-

CAMP (0.15 mM) were 1807.7 * 120.0 pg/ml, 945.7 * 188.4 pg/rnl, and 136 1.5 102.6

pg/ml, respectively. Values represent the mean k SEM of 2 to 4 fish with 4 replicate

incubations per treatment. Astensks represent T levels that are significantly lower than

controls (Split-Plot ANOVA, p< 0.05).

+ TNF

Fig. 2.14. Effect of tumor necrosis factor a (TNFa) on the conversion of steroid precunon to

testosterone (T). Testis pieces were incubated with 25-hydroxycholesterol (250H-chol; 1 pglml),

75OH-chol ( 1 O &ml), pregnenolone (50 @ml), pregnenolone (1 00 nghl), and 17a-

hydroxyprogesterone (1 7aOH-progesterone; 50 ng/ml) with and without TNFa (IO ng/ml).

T estis pieces were incubated for 1 8 hr at 18 " C. Values represent the mean k SEM of 2 fish with 4

repiicate incubations per treatment. In the same order as listed above, the leveis of T for the

control groups were 436.4 * 22.4 pg/ml, 1 145.6 + 49.1 pg/ml, 14 19.5 * 8 1.7 pg/ml, 24 10.3 * 270.7 pg/ml, and 1050.6 * 59.0 pg/ml. Asterisks represent significant differences compared to

incubations without TMa (Split-Plot ANOVA, p< 0.05).

T Ci Control

Fig. 2.15. Effect of tumor necrosis factor a (TNFa) on testosterone (T) production by

testis pieces stimulated with hCG (0.2 iüfml) and 25-hydroxycholesterol(250H-chol; 1

and 10 pglrnl) combined. Testis pieces were incubated for 18 hr at 18°C. The production

of T after treatment with hCG + 25OH-Chol(1 pg) and hCG + 250H-Cho[( IOpg) was

2680.2 * 224.3 pg/ml and 3017.7 * 432.3 pg/ml, respectively. Values represent the mean

i SEM of 2 fish with 4 replicate incubations per treatment. Asterisks represent T levels

that are significantly different cornpared to controls (Split-Plot ANOVA, p< 0.05).

DISCUSSION

Although the multifactorial regulation of testicular steroidogenesis is described

well in mammals (for reviews refer to Saez, 1994; Gnessi et al., 1997), the present çtudy

contributes ro the reiatively small, but progressively increasing field involved with the

paracrine/autocrine regulation of testicular functioning in teleosts. The results of this

study suggest that the role of "immune-denved" peptides in testicular steroidogenesis is a

phenornenon that is conserved between mammals and lower vertebrates.

The inhibition of gonadotropin-stimulated androgen production by mamrnalian

TNFa and IL- 1 P, and by macrophage-derived products in the goldfish testis, parallels

numerous studies in marnmals that have documented the effects of cytokines on testicular

function (reviewed in Hales, 1996; Hales, 2000). Generally, the reported effects of TNFa

on basal T production are more variable. In the present study, a potentiating effect of

TNFa was observed on basal T production of testis that was either resuming

spematogenesis or was in a regressed state, as indicated by gross morphology and the

GSI value. In contrast, TNFa clearly inhibited T production of spermiating testes. The

diametric response of the tissue to TNFa on basal T production suggests that the action of

this cytokine varies during the reproductive cycle in teleosts, possibly influencing aspects

of testicular deveiopment. The potentiating activity of TNFa on basal T observed in the

present study coincides with only one report on adult rat Leydig cells (Warren et ai.,

1990), while other mamrnalian studies have documented inhibitory effects of TNFa on

basal adult mouse (Xiong and Hales, 1993b; Xiong and Hales, 1997) and no effect on

basai immature porcine (Mauduit et al., 199 1 ; Mauduit et al., 1998) Leydig cell androgen

production. The reasons for these discrepancies are uncertain but may involve differences

in methodologies, species-specificity, as well as the matunty of the ceil preparations.

Typically, mammalian studies have not examined the effects of cytokines on Leydig

steroidogenesis of both immature and mature animals under the same laboratory

conditions. However, support for the involvement of cytokines in normal developrnent is

eenerated frorn the studies of Khan et al. (1992a; 1992b) that indicate that IL- lP, IL- la , Y

TNFa, and TGFa stimulate immature Leydig ce11 proliferation and differentiation but do

not affect adult Leydig ce11 DNA synthesis.

In the present study, the inhibition of basal T production by IL- I P is not consistent

with several studies conducted on rat (Verhoeven et al., 1988; Warren et al., 1990) and

mouse (Xiong and Hales, 1998) purified or dispersed Leydig crlls that report either

stimulation or no effect of IL-1P on basai T levels. It is unlikely that this dissimilarity is

due to the dose of IL-IB as the doses used in this study are similar to the other reports.

The contrasting results may be owing io the whole tissue system employed in the present

study, whereby the cytokines and macrophage-derived products could exert their effects

on T production indirectly through other testicular cells. The maximal degree of

inhibition observed throughout the present study tended to be less drastic than similar

studies ernploying Leydig cell cultures and may also be accredited to the use of testis

pieces instead of purified Leydig cells.

Cytokines exhibit the characteristics of redundancy, pleiotrophy, synergy, and

antagonisrn, which facilitates their interactive and coordinated regulation of cellular

activities (Kuby, 1994). TNFa and IL-1P have been previously reported to have either

synergistic effects (Lin et al.. 1994) or additive effects (Calkins, et al., 1990) on

mammalian Leydig ce11 steroid production. In the ûpresent study, the effect of the

combined treatment of TNFa and IL- 1 fl appeared to be additive, yet, due to the non-

drastic maximal reduction in T levels and the consemative nature of the statisticai test

used, this effect was not found to be significant.

In the present study, supematants from the RTS 1 1 macrophage ce11 line were

employed in the testis incubations as a possible source of fish-specific cytokines.

Ganassin and Bols (1998) believe RTS 1 1, which is composed of large, granular

macrophages and smaller, round cells, to secrete active cytokines or growth factors as

RTS 1 1 supematants enhance their own growth, as well as induce proliferation of trout

head kidney leucocytes. The effect of the RTS 1 1 supernatants on goldfish T production

are consistent with the studies of Sun et al. (1993) and Sun and Risbridger ( 1994) that

found that rat testicular macrophage conditioned media (TMCM) inhibited LH-stimulated

T production but had no effect on basal levels. However, these results are in contrast to a

previous report by Yee and Hutson (1985) that demonstrated a stimulatory effect of rat

TMCM on both basal and LH-stirnulated Leydig cells. The majority of the studies

describe inhibitory effects of MCM on gonadotropidcAMP-stimulated androgen

production and either an inhibition or no effect on basal T levels (reviewed in Hales,

1996). Possible reasons for the discrepancies in the results may involve the different

densities of the macrophage cultures, as well as the heightened sensitivity to extemal

stimuli by macrophages (Moore and Hutson, 1994). The biphasic effect of the RTS 1 1

supematants observed in the present study is also supported by the reports of Sun et al.

( 1 993) and Yee and Hutson (1985). MCM appears to be inhibitory at low concentrations

41

but stimulatory or have no effect at higher concentrations.

It is difficult to know with certainty, whether or not the RTS 1 1 supematants used

in this study contain IL- 1 - or TNF-like factors. Westem blot analyses of RTS 1 1

supematants with marnmalian antibodies against mouse TNFa and IL- 1 P revealed

proteins in supematants From LPS-stimulated cells only (refer to Chapter 3). In spite of

these results, a highly sensitive mouse TNFa ELISA failed to detect TNF-like factors in

unstirnulated and stimulated RTS 1 1 supematants (data not shown). Similarly, the

inability to nullib the activity of the RTS 1 I supematants through the use of marnmalian

TYFa and IL- I fl antibodies does not provide definitive proof that the active factor(s) was

not RIF-like andor IL- l-like proteins. The inconsistent results may be inherent to my

studies as the results depend on the cross-reactivity of molecules of different classes

(mammals to fish). To successfblly nulliEj the activity of the conditioned media, the

mamrnalian TNFa and IL- 1 P antibodies employed would be required to bind the putative

fish cytokines in such a way as to inhibit their activity. It is plausible in this experiment

that the antibodies may have bound the proteins without interfenng with their ability to

modulate steroidogenesis, or may have failed to bind the active proteins altogether.

Confounding the results, were the unexpected decreases in hCG-stimulated T levels by

the mammalian antibody control treatrnents while non-immunized sera did not influence

T production. The reason(s) for this decline in T production is unknown. It is

conceivable that these irnrnunoglobulins could bind endogenous factors in the testis

incubations and prevent their activities or their interactions with other molecules which

might result in decreased T production through an unknown mechanism. In other studies,

the activity of stirnulated fish immune ce11 supematants was blocked by antibodies against

rnamrnalian IL- la and IL- 1 P (Ellsaesser and Clem, 1994; Verburg-van Kemenade et al.,

1995) and a monoclonal antibody against the mammalian 55 kDa TNF receptor (TNF-RI)

(Jang et al., 1995a).

The activities of mammalian TNFa in the goldfish testis incubations closely

resemble its activities documented in the mammalian literature where the plieotropic

nature of this cytokine is illustrated well by its multiple sites of action in the Leydig ce11

steroid biosynthetic pathway (reviewed in Hales, 2000). Although TNFa has been

reported to act both proximal to CAMP activity through the inhibition of gonadotropin

binding and cAMP formation (Calkins et al., 1990; Mauduit et al., 199 l ) , the majority of

mammalian studies indicate that its predominant site of action is distal to cAMP via the

inhibition of key steroidogenic enzymes (reviewed in Hales, 2000). In contrast to the

reports of Calkins et al. (1990) and Mauduit et al. (199 l), basal and hCG-induced CAMP

formation was not affected by TNFa treatment in the goldfish testis incubations.

However, the inhibitory effects of TNFa on hCG-, fonkolin-, and 8-bromo-CAMP-

stimulated T production in the present study coincide with the results in porcine (Mauduit

et a1.,1991), mice (Xiong and Hales, 1993b), and rat (Lin et al., 1994; Li et al., 1995)

Leydig ce11 cultures. The cytokine induced a small inhibitory effect on pregnenolone-

stimulated T levels but only at the higher dose tested (100 ng/ml) and did not affect 17a-

hydroxyprogesterone-stimulated T levels. Combined, these results indicate that a major

site of ï N F a activity in the steroid pathway involves the mobilization of cholesterol or its

availability to the mitochondria and to a lesser degree, TNFa may affect the 3B-

hydroxysteroid dehydrogenous (3PHSD) enzyme responsible for the conversion of

pregnenolone to progesterone. A previous report by Xiong and Hales (1997) found that

TNFa reduced basal and CAMP-stirnulated 3PHSD expression in mouse Leydig cells.

Steroid hormone biosynthesis requires both the delivery of cholesterol to the

mitchondria outer membrane and the de novo synthesis of the steroidogenic acute

regulatory (StAR) protein to facilitate cholesterol translocation to the inner mitochondrial

membrane (Thomson, 1998). The disappearance of the inhibitory effect of TNFa on T

production stimulated with

250H-ch01 in the present study suggests that TNFa might affect steroid synthesis at the

lcvel of cholesterol delivery to the inner mitochondrial membrane. Recently, a rainbow

trout StAR, with 55% arnino acid similarity to mammals, was found to be expressed in

trout steroidogenic tissues, inciuding the testis (Todo et al., 2000). In porcine (Mauduit et

al., 1998) and MA- 10 mouse tumor (Budnik et al., 1999) Leydig cells, TNFa decreased

both StAR gene expression and protein synthesis making this a potential site of TNFa

activity in goldfish testis.

In surnmary, the present study provides evidence for the involvement of the

proinfiammatory cytokines, TNFa and IL- 1 P, and possibly other macrophage-derived

products, in the regdation of goldfish testicular steroid biosynthesis. The cytokines and

RTS 1 I macrophage supematants resulted in the inhibition of gonadotropin-stimulated T

production but affected basal T more variably. Further work is required to identiv the

factor@) in the macrophage supematants that is responsible for the inhibitory effect on

gonadotropin-stimulated testosterone production. The mechanism by which TNFa acts in C

teleost gonads resembles its activities in mammalian Leydig ceIl cultures and appears to

affect the availability of cholesterol, as well as enzymes in the steroid pathway. The

results of this study suggest that the role of cytokines in testicular steroidogenesis is a

phenornenon that is conserved between mamrnals and lower vertebrates. Further work is

warranted to elucidate the roles and relevance of cytokines in fish reproduction under

normal physiological states. as well as in immune-activated conditions.

CHAPTER 3

Modulation of Testosterone Production in Male GoIdfish and Induction of

Cytokine-like Molecules in a Trout Macrophage Ce11 Line by the Immune

Stimulant, LPS

INTRODUCTION

It is weli established in higher vertebrates that inflammation and immune

dysfunctions have deleterious effects on reproductive processes in males.

Lipopolysaccharides (LPS: bacterial endotoxin) account for many of the disease

symptoms of Gram-negative bacteria infection. Primarily, the mechanism of LPS activity

is not direct but through the induction of mediators such as nitric oxide, prostaglandins,

and pro-inflammatory cytokines (e.g. IL- 1, TNFa, IL-6, IL-8) from activated monocytes

and macrophages (Fùetschel and Brade, 1992; Sweet and Hume, 1996). The binding of

LPS to its receptor, CD 14, on the surface of macrophages is mediated via plasma binding

proteins (LPS-binding proteins) and activates several signal transduction pathways

(reviewed in Sweet and Hume, 1996).

Several studies have observed inhibition of testicular steroidogenesis and

disruption of spermatogenesis in mammals treated with LPS (Christeff et al., 1992;

Wallgren et al., 1993; O'Bryan et al., 2000a). In vivo, LPS may compromise testicular

Function at multiple levels through the activation of the hypothalamic-pituitary-adrenal

(H-P-A) axis (elevated glucocorticoids) (Hales et al., 2000) and inhibition of the

hypothalamic-pituitary-gonadal (H-P-G) axis (decreased GnRH and LH release) (Takao

et al., 1993; O'Bryan et al., 2000a). However, inhibition of Leydig ce11 function at the

testicular level also occurs during infiammatory conditions and is responsible for the

decline in androgen levels (O'Bryan et al., 2000a). Testicular LPS-induced cytokines,

such as TNFa and IL4 P, are known effectors of steroidogenesis and have been

implicated as regulatory molecules of reproductive processes during both normal and

inflammatory conditions (reviewed in Hales, 2000).

In teleosts, the bidirectional communication between the immune and endocrine

systems is primarily described in the context of aquaculture and its associated stressors

( e s . crowding, iransportation) that activate of the hypothalamic-pituitary-interrenal (H-P-

1) axis (reviewed in Balm 1997 and Weyts et al., 1999). However, there is a paucity of

information regarding the concerted actions between immune components and the H-P-G

axis in teleosts. One report by Loir et al. (1995) discerned that factors secreted by trout

testicular macrophages were both stimulatory and inhibitory on DNA synthesis in

spermatogonia and spermatocytes depending on the macrophage preparation. In addition

to the knowledge that fish are relatively insensitive to the toxic effects of LPS compared

to rnammals (White and Fletcher, 1985), it has been demonstrated that LPS

administration into tilapia results in a vanety of physiological changes including

stimulation of the interrenal axis (increased cortisol levels) and increased epidermal

thickness and opercular chloride ce11 numben (Balm et al., 1995). Currently, there are no

reports that directly examine the effects of LPS on teleost reproductive physiology and 1

postdate that teleost gonadal steroidogenesis will not escape the modulating effects of

LPS given the described mammalian responses and the homology that exists between the

steroid biosynthetic pathways of marnmals and teleosts.

To substantiate the fundamental impact of immune activation on testicular

endocrinology in teleosts, 1 investigated the effects of LPS administration in vivo on

circulating plasma T levels in goldfish, as well as the effects of LPS on T secretion of

testis pieces in vitro. In marnmals, one mechanism by which LPS modulates gonadal

steroidogenesis is via the induction of pro-inflammatory cytokines from leukocytes

residing within the testes. Cut-rently, progress in the research of IL- 1 and R I F in fish is

npid and both genes have recently been sequenced. Therefore, we examined the

potential for teleost leukocytes to secrete IL- Land RIF-like peptides in response to LPS

by conducting Western blots of LPS-stimulated trout macrophage supematants from the

RTS 1 1 ce11 line. We also investigated the endogenous production of these cytokines by

gold fish kidney and testis.

MATEEU.ALS AND METHODS

Fislz

Common goldfish were purchased from DAP International (Etobicoke, ON) and

were maintained at the Hagen Aqualab, University of Guelph, in 1.8 rn diameter circular

tanks with flow through water at 16- 18 ' C under a constant photopenod ( 14 hr light/ 1 O hr

dark). Fish were fed once daily to satiation.

Ch emicals, Hormones and A ntibodies

Human chorionic gonadotropin (hCG) and lipopolysaccharide (LPS, E. Coli

serotype 026:B6) were purchased From Sigma Chemical Co. (St. Louis, MO). Leibovitz

L-15 media with L-glutamine, penicillin, streptornycin were purchased from Gibco BRL

(Burlington, ON). Murine recombinant turnor necrosis factor a ( m a ) and interleukin-

48

I B (IL- 1 B). and affinity purified polyclonal rabbit anti- mouse TNFa antibody were

purchased from Chernicon International, Inc. (Temecula, CA). The polyclonal rabbit

anti-human IL- 1 P antibody was bought h m Upstate Biotechnology (Lake Placid, NY).

Stock solutions of LPS were dissolved directly in L- 15 media or sterile filtered

phosphate buffered saline (PBS, pH 7.5) and vortexed for 15 min to allow the LPS to go

into solution before storage at 4°C.

Testis Incubations

Goldfish testis incubation procedurc was modified from Wade and Van Der Kraak

( 199 1) and was previously described in Chapter 2, Materials and Methods. The

gonadosomatic index (GSI; GSI = gonad weight/ (body weight-gonad weight) X 100)

range o f the goldfish used in the in vifro incubations was 3.4-7% (average 4.2% * 0.5).

I n Vivo LPS Adrninistratioti

This experiment was conducted twice and samples were later combined to

increase the respective treatment groups. Expenmental procedures were identical

between the hvo trials. Male goldfish were chosen for the study based on 1) the strong

presence of the secondary sexual characteristic, tubercles, and 2) possible remnant

tubercles. Fish were randomly distributed into 2 experimental groups: a control group

(first expenment n=l 1, second expenment n=l 1) and a LPS-treated group (first

experiment n=l 1, second experiment n=9). Each group of fish was held in 0.7 m through

flow tanks for the durarion of the experiment and were allowed to acclimate to the new

tanks for 3 days. Photoperiod, temperature and feeding were the same as holding

conditions, except that fish were not fed on the last day of the expenrnent. AAer the

acclimation period, goldfish were anaethetized with MS222, weighed, and injected IP

with 500 pg LPS in 250 pl sterile PBSISO g body weight (Day 1). Control fish were

injected with sterile PBS at 250 pV50 g body weight. On Day 4, al1 fish were

anaethetized with MS222, weighed, and blood was taken by caudal vein puncture into

heparinized synnges. Blood samples were spun at 3000g for 10 min and plasma T was

extracted by the method of McMaster et al. (1992). Goldfish were killed by cervical

transection and testes and kidneys were removed, weighed, and snap frozen in liquid

nitrogen and stored at -80°C to be later used in Western blot analyses. No fish died

during the experiment. The final sample size for controls and LPS-injected fish was 17

and 1 5, respec tively .

R TSl I Supernatant Preparation

RTS 1 1 cells (Dr. N. Bols, University of Waterloo; Ganassin and Bols, 1998) were

maintained in continuous culture at the University of Guelph in L-15 medium

supplemented with 25% fetal bovine serurn and penicillin/streptomycin at 18°C without

supplemental CO,. P ior to use, cells were harvested from tissue culture flasks by gentle

scraping and centrifuged at 200g for 5 min. The supematant was aspirated and replaced

with serum-free L- 15 media. CelIs were then cuhred at 500 000 cells/ml in 24 well

plates at 18°C. Cells were allowed to adhere for 2 hr. Next, 100 pl of media was

removed from a11 of the wells and replaced with 100 pl of fresh media containing 20

pplml LPS to stimulate the cells. The LPS was previously dissolved directly into L- 15

media. The cells were incubated for 24 and 48 hr. Control wells were treated similarly,

but LPS was ornitted. After the incubation penods, the RTS I 1 macrophage conditioned

media (MCM) was collected, spun for 5 min at 2009 to pellet any debris, and

concentrated using Centriprep 10 (1 0 kDa molecular weight cut-off) centrifuga1 filter

devices (Millipore Corp., Bedford, MA). Samples were concentrated approxirnately 10

fold and snap frozen in liquid nitrogen and stored at -80°C to be later used in Western

blot analyses. Protein concentrations were deterrnined for each treatment by DC Protein

assay (Bio-Rad Laboratories, Missisauga, ON).

Tissue Preparation

Goldfish testes and kidneys frorn the in vivo LPS injection experiment were

placed in ice cold lysis buffer consisting of 50 rnM Tris-HC1 (pH 7 . 3 , 150 mM sodium

chloride, 1% Triton-X 100,0.5% sodium deoxycholate, Pefabloc SC (O. 1 mghl ;

Boehringer Mannheim, Laval, PQ), trypsin inhibitor (20 pig/ml) and leupeptin (4.25

pg/ml) (Sigma Chemicai Co., St. Louis, MO). Kidneys were used in the analysis for

cytokines because the anterior kidney (head kidney) is the pnmary site of haematopoiesis

and immune system function in fish, therefore, it would be expected that the kidneys

would be a significant source of cytokines. Tissues were briefly homogenized by 3

passages using a Potter-Elvehjem motor-dnven Teflon pestle. Homogenates were

centrifuged at 4OC for 5 min at 2000g to pellet debris and then the supernatant was

collected and centrifùged for 30 min ai 15000g. This supernatant was collected and

concentrated approximately 10 fold using Centnprep 10 devices. The concentrated

fraction was snap fiozen in liquid nitrogen and stored at -80°C. The amount of protein in

each sample was quantified using the DC Protein assay. These samples were used in

Western analyses or subjected to TNFa immunoprecipitation (TP) followed by Western

analyses. Immunoprecipitation of the homogenates was the same as descnbed by the

manufacturer of the Protein-A-Agarose (Boehringer Mannheim, Laval, PQ). The

concentratrd homogenates were standardized to 10 mg of protein and rnixed with anti-

murine TNFa antibody (3 pg) for 3 hours at 4 O C . Immunoprecipitates were isolated with

protein-A-coupled agarose and carefully washed several times. Control

immunoprecipitation reactions included marine TNFa ( 100 ng) rnixed in lysis buffer and

homogenates rnixed with non-immune rabbit sera (no pnmary antibody).

Immunoprecipitates were used immediately or frozen and stored at -20°C pnor to being

used in Western blot analyses.

TNF a and IL- I f l Western Blots

Proteins in the RTS 1 I supematants ( 10 pg), tissue homogenates ( 100 pg), and

irnrnunoprecipitates were resolved by 12% SDS-polyacrylamide gel electrophoresis for

approximately 1 hr at 150 V. Positive controls included munne TNFa. (1 0 ng), murine

IL-1 P (50-75 ng) and an immunoprecipitated sarnple of munne TNFa. L- 15 media or

lysis buffer served as the appropriate negative control. Proteins were transferred

ovemight at 29 V onto a PVDF membrane (Bio-Rad Laboratones, Laval, PQ). Blots

were blocked for 1 hr in PBS-Tween, containing 5% non-fat dry rnilk (w/v) with

constant shaking. The presence of TNF-like and IL- 1 -1ike proteins were detected using

rabbit anti-rnouse TNFa (0.3 @ml) and rabbit anti-human IL-1P (1 pg/ml) as pnrnary

antibodies (1 hr incubation at room temperature), respectively. This was followed by 1 hr

incubation with goat anti-rabbit IgG conjugated with honeradish peroxidase as a

secondary antibody (0.3 pglml). Incubation of the membranes with secondary antibody

alone. as well as non-immune rabbit semm (naive IgG; 0.3 @ml), were routinely

included to determine potential non-specific binding. Immunoreactive bands were

visual ized by the ECL Western blotting system ( h e r s h a m Pharmacia, IL).

Statistical Analyses and Presentation of Data

T levels of repeated expenments were converted to % of hCG-stimulated T

production for graphing and statistical purposes. The differences between treatment were

determined by the General Linear Models procedure (proc GLM; SAS Institute Inc.,

Cary, NC) and when warranted, this was followed by Tukey's HSD post hoc test. A p

value of ~ 0 . 0 5 was considered significant. Differences in treatment means of plasma T

levels in the LPS injection expenment were compared using the non-parametric Mann-

Whitney test statistic. Levels of plasma T that were below the detection limit of the N A

were assigned the detection limit value (1 S6 pg) as this is the rnost consemative estimate.

RESOLTS

Effects of L PS on basal and h CG-stim itlated testosterone production

As s h o w in Fig. 3.1, LPS tended to potentiate basal T production (p=O.O 12) of

goldfish testis in vitro. The highest dose of LPS tested (10 pgiml) resulted in a 9 1%

increase in T levels compared to the control group. Conversely, Fig. 3.2A shows that

treatment with LPS caused a significant and dose-dependent decrease in hCG-stimulated

T production. Testosterone was decreased by 26% and 50% with 0.2 and 20 pg/ml LPS

(p=0.007), respectively. LPS at 2 @ml inhibited gonadotropin-stirnulated T production

(Fig. 3.2B) with an average inhibition of 3 1% (p=0.000 1).

Effects of LPS on testosterone production in vivo

The male goldfish used in this study exhibited a wide range of GSI values within

each treatment group. As the levels of circulating androgens in teleosts depend on the

reproductive stage of the gonad, the fish in this study were divided into 2 groups based on

their GSI value (GSI 4 . 0 and GSI >1.0) in order to reduce the variability of plasma T

levels within treatments. Sample sizes permitted the creation of only hvo groups. Table

3.1 demonstrates the considerable differences in GSI values between the designated

groups and the average GSI value of fish within each treatment. Injection of LPS into

male goldfish (n=8) causes an inhibition of testosterone production compared with

controls (n=9) in fish with a GSI ~ 1 . 0 (Fig. 3.3A). Fie. 3.3B indicates that LPS injection

of fish (n=7) in a more advanced stage of the spermatogenetic cycle (GSI > 1 .O) did not

have an rffect on plasma T Ievels cornpared with controls (n=8) but these T levels were

highly variable. In the G S I 4 .O LPS-treatrnent g~oup, 4 of 8 goldfish had non-detectable

levels of plasma T. No other fish in the study showed non-detectable levels of T.

Western b fotting of R TSl 1 superna tants

Since LPS is known for its ability to stimulate macrophages and upregulate

cytokine production, Westem blotting was used to determine if the RTS 1 1 cells are

responsive to LPS and subsequently produce identifiable TNF- or IL- 1 -1ike proteins.

Westem blots with anti-rnouse TNFa of concentrated RTS 1 1 supernatants (10 pg of

protein) collected fiom 48 hr ce11 cultures that were treated with LPS revealed

immunoreactive bands of approximately 17.1 and 15.8 kDa (Fig. 3.4A, Lane 5). A

different LPS-treated RTS 1 1 supernatant sample (Fig. 3.4B) that was prepared in the

same manner as Fig. 3.4A demonstrates 3 visible bands of approxirnately 18.3, 17.4, and

16.2 kDa (Lane 5). Similar bands were barely visible from supematants prepared from

24 hr LPS-treated ce11 cultures (data not shown) and no bands were visible fiorn control

ce11 culture supernatants of either tirne period. Mouse TNFa, run concurrently as a

positive control, displayed one band at 17.3 D a (A) and 17.2 kDa (B). Western blot

analyses with a rabbit anti-human IL-1 P antibody of concentrated RTS 1 1 supernatants

(Fig.3.5; 1 O pg of protein) yielded 2 higher molecular weight species of approximately

27.7 and 24.0 kDa, as well as a prominent band at 15.6 kDa. Sirnilar bands were

visualized with supematants generated from 24 hr ce11 cultures (data not shown).

Multiple bands between approximately 16-17 kDa are also evident on this blot.

Incubation of the membrane with secondary antibody (goat anti-rabbit IgG) and non-

immune rabbit semm (a source of "non-immunized" nbbit IgG) did not reveal any bands

(data not shown).

Testes and kidney homogenates (100 pg protein each) of fish injected with LPS in

vivo for 72 hr were immunobIotted with rabbit anti-mouse TNFa and rabbit anti-human

IL- 1 P (Fig. 3.6). The positive controls, mouse TNFa (IO ng) and mouse IL4 B (50 ng),

were visuaiized as single bands at molecular weights sirnilar to the known molecular

weights of these peptides. These biots are void of other immunoreactive proteins.

lmrnunoprecipitation of the same sarnples (above) with the primary antibody to rnouse

TNFa did not reveal TNF-like bands. There were no differences noted between the

kidney and testis homogenates of either control or LPS exposed fish. The expected heavy

and light chains of the mouse IgG molecules used in the immunoprecipitation reaction

(approximately 45 and 26 D a ) (Fig. 3.7.) were visualized. The control lane shows that

mouse TNFa was precipitated out of the lysis buffer at the appropriate molecule weight.

Because several faint bands are identifiable both in the control (Fig. 3.7.A) which did not

contain sample homogenate and in the homogenates (Fig. 3.7B. lanes 4-7), these multiple

bands are thought to be fragments of the rabbit IgG molecules used in the

immunoprecipitation reaction. This was further supported by their presence on blots of

similar membranes that were incubated with only secondary antibody during the Westem

procedure. To determine non-specific binding by the pnrnary antibody to non-TNF-like

proteins, control reactions of sarnple homogenates were immunoprecipitated with non-

immune rabbit serum instead of pnrnary antibody (Fig. 3.7B, lanes 9-10). Multiple faint

bands. as well as the expected heavy and light chain IgG molecules, were visualized in

lanes 9 and 10. Also, incubation of membranes that consisted of immunoprecipitation

reactions of tissue homogenates with secondary antibody alone dunng the Westem

procedure (data not shown) revealed the presence of heavy and light IgG molecules, as

well as several other similar bands.

LPS (pgl ml)

Fig. 3.1. Effect of graded doses of E. Coli lipopolysaccharide (LPS; 0.08-10 pglml) on

basal testosterone production from testis pieces incubated for 1 8 hr at 18 O C . Values

represent the mean * SEM of 1 fish with 4 replicate incubations per treatrnent. Values

designated by the same letter are not significantly different fiom each other (Tukey's test,

p< 0.05).

Fig. 3.2. Effect of A) low (0.2 pg/rnl) and high (20 pg/rnl) doses and B) 2 pgiml of

lipopolysaccharide (LPS) on hCG (0.2 1Ulrnl)-stimulated testosterone (T) production from testis

pieces incubated for 18 hr at 18°C. In B) the levei of T in the control gmup was 2578.6 239.6

pg/rnl. Values designated by the same lener are not significantly different from each other (A)

Tukey's test and B) Split-Plot ANOVA, p c 0.05).

Table 3.1. A description of the average gonadosomatic index (GSI) ranges of male

goldfish lrom Fig. 3.3 injected with lipopolysaccharide (LPS; 500 @ml) or phosphate

buffered saline (carrier). Values shown are the average GS1 of the designated treatment

group SEM of A) control n= 9; LPS n= 8 and B) control n= 8; LPS n= 7. There are no

significant differences in GSI values between controls and LPS-injected goldfish within

the GSI ranges given (ANOVA, p< 0.05).

CONTROL

LPS-INJECTED

A) GSIc1 .0

0.49 * 0.07 0.52 0.07

B) GSI> 1.0

1.97 k 0.31

2.84 * 0.42

Control LPS Control WS

Fig. 3.3. Plasma testosterone (T) levels in control and E. Coli Iipopolysaccharide (LPS)-

injected male goldfish with A) a gonadosomatic index (GSI)<l .O and B) a GSbl.0. The

LPS group received a single IP injection of LPS at 500 pg/50 g body weight and controls

were injected with phosphate buffered saline. Al1 fish were killed 72 hr after injection.

A) control n= 9; LPS n= 8. B) control n= 8; LPS n= 7. Values represent the mean * SEM of each treatment group. Astensk represents T levels that are significantly lower

than conirois (Mann-Whitney U test, p 0.05).

Fig. 3.4. SDS-PAGE and Western blot analyses with rabbit anti-mouse turnor necrosis

factor a (TNFa) of proteins in concentrated RTS 1 1 supematants generated from 48 hr

ce11 cultures with and without E. Coli lipopolysaccharide (LPS) (20 pg/ml). The Western

blots (A and B) are of separate sarnples prepared as described in Materials and Methods.

Lanes: 1) L- 15 media (negative control), 2) marker proteins, 3) mouse TNFa ( 10 ng,

positive control), 4) control RTS 1 1 ce11 culture supematants, 5) LPS-treated RTS I 1 ce11

culture supematants.

Fig. 3.5. SDS-PAGE and Western blot analysis with rabbit anti-human interleukin- 1 P

(IL- 1 P) of proteins in concentrated RTS 1 1 supernatants generated from 48 hr ce11 cultures

with and without lipopolysaccharide (LPS; 20 pg/ml). Sarnples prepared as described in

Materiais and Methods. Lanes: 1 ) L-15 media (negative control), 2) marker proteins, 3)

mouse IL- 1 P ( 100 ng, positive control), 4) mouse IL4 P (50 ng, positive control), 5)

control RTS 1 1 cell culture supematants, 6) LPS-treated RTS 1 1 ce11 culture supematants.

Fig. 3. 6. SDS-PAGE and Western blot analysis of A) anti-mouse tumor necrosis factor a

(TNFa) antibody and B) anti-hurnan interleukin-1 p (IL-1 P) antibody of testis and kidney

homogenates of fish used in Fig. 3.3. Samples in Ianes of A) are: 1) buffer, 2) marker proteins, 3)

mouse MFa (10 ng), 4 and 5) gonadosomatic index (GSI)>l .O control and lipopolysccharide

(LPS)-injected testis homogenate, 6 and 7) GSkI .O control and LPS-injecred testis homogenate, 8

and 9) G S P 1 .O control and LPS-injected kidney homogenate. Samples in lanes of B) are: 1)

marker proteins, 2) mouse IL-lB (50 ng), 3 and 4) GSI>l.O LPS-injected testis and kidney

homogenate, and 5) buffer. Samples were prepared as described in Matenals and Methods.

TNFa

Fig. 3.7. Representation of SDS-PAGE and Western blot analysis of proteins from testis

and kidney hornogenates frorn control and lipopolysaccharide (LPS)-injected goldfish.

Homogenates were immunoprecipitated with an anti-mouse tumor necrosis factor a

(TNFa) antibody and blotted with the same pnmary antibody. Control lane (A) is mouse

TNFu immunoprecipitated with anti-mouse TNFa. In B) lanes are: 1) buffer, 2) marker

proteins, 3) mouse TNFa (long), 4 and 5) immunoprecipitated control and LPS-injected

testis homogenates, 6 and 7) immunoprecipitated controol and LPS-injected kidney

homogenates, 8) empty, and 9 and 10) LPS-injected testis and kidney homogenates mixed

with non-immune rabbit sera and immunoprecipitated.

DISCUSSION

The present study, to my knowledge, is the first to demonstrate that the immune-

stimulant, LPS, affects the levels of testosterone in fish. Plasma T levels were reduced in

goldfish injected with LPS in the GSI< I .O treatment group and hCG-stimulated T levels

of testis pieces were inhibited Ni vitro by LPS in a dose-dependent manner. Basal levels

of T by goldfish testis appeared to be stimulated by LPS exposure. These results (and the

results of Chapter 2) support the growing evidence of complex communication between

the immune and endocrine systems in teleosts; a hypothesis that is relatively unexplored

in fish but widely described in the marnmalian literature. The results of this study also

indicate that bactenal endotoxin administration may be appropriate for future

investigations of immune-endocrine interactions in teleosts that focus on events

associated with the H-P-G a i s , as has been the case for studies examining the effects of

stress and immune activation on the H-P-I axis (Balm et al., 1995; Weyts et al., 1999).

The decline in T levels in goldfish injected IP with LPS is consistent with

mammalian studies showing that LPS injection into rats (Christeff et al.. 1987; Chnsteff

et al., 1991), mice (O'Bryan et al., 2000b) and boars (Wailgren et al., 1993) results in

decreased circulating androgens. Remarkably, a single IP injection of LPS (200 pg) into

mice caused a rapid decline in testosterone production (within 2hr) that was sustained for

several days (Bosmann et al., 1996). In consideration of the dose of LPS used in the

present in vivo study (10 mgkg body weight) compared with the previous mammalian

studies (dose range 0.1-8 mgkg), it is necessary to note that fish are known to be more

tolerant to LPS and exhibit a much higher LD, compared with rnammals (Kodarna et al.,

1987). Numerous inoculation studies on fish have described single to multiple injections

of known fish pathogen endotoxins and commercially available E. Coli LPS at similar

doses to the one used in my study (MacArthur et al.. 1984; Al-Harbi and Austin, 1992).

For example, tilapia (300g) were injected with 3mg/kg E. Coli LPS 4 times, every other

day, in studies by Balm et al. (1995) that examined a variety of physiological endpoints.

There are several possible mechanisms by which LPS may be acting to modulate

in vivo levels of T in the GSKI.0 treatment group in the present study. Endotoxin

exposure stimulates adrenal (interrenal) steroidogenesis in both mammals (Rivier and

Rivest, 1991) and fish (Balm et al., 1995; Balm, 1997; Weyts et al., 1999) which results in

elevated levels of glucocorticoids. In mammals, elevated glucocorticoids are known to

inhibit the H-P-G mis at multiple sites, including GnRH and gonadotropin secretion. and

to exert direct inhibitory effects on Leydig cells (Rivier and Rivest, 1991). Increased

circulating levels of cortisol in fis h are associated with reproductive impairments

including depressed plasma T and estradio1 levels, reduced gonad size, and decreased

plasma GtH levels (reviewed in Pankhurst and Van Der Kraak, 1997), as well as

increased disease susceptibility (Balm, 1 997). Therefore. it can be hypothesized that in

the present study, LPS injection into goldfish depressed plasma T levels through the

activation of the H-P-1 mis, possibly mediated through the activities of cytokines. Direct

evidence of cytokine-mediated activities in fish is lacking, however, fish leukocytes

exposed to LPS (and other rnitogens) have been s h o w to secrete cytokine-like molecules

(Ellsaesser and Clem, 1994; Verburg-van Kemenade et al., 1995; Neumann et al., 2000).

In fish, the head kidney supports the production of cortisol, haematopoiesis, and antibody

production, which would allow direct paracrine interactions to occur between the immune

66

and endocrine systems (Weyts et al., 1999). Recombinant trout IL- 1 P injected IF into

trout elevates plasma cortisol levels which provides tùrther support for the involvement of

cytokines in the activation of the H-P-1 mis in fish (Holland et al., 2000).

Another possible mechanism by which LPS reduced T levels in the in vivo, as well

as the in vitro experiments in this study involves the inhibition of steroidogenic enzymes

and/or St4R proteins within the testes. In marnmals, the inhibition of Leydig ce11

steroidogenesis during experimental endotoxemia has been found to occur pnmarily at the

level of the testes. O'Bryan et al. (2000a) and Hales et al. (2000) found that T levels are

rapidly reduced in rnice after a single injection of LPS, and that this effect was present

even though LH levels remained unchanged. The long term inhibition of testosterone

production observed after LPS administration (6 hr to 9 days) in mice was Iikely due to

cytokine-suppressed steroidogenic enzyme gene expression of' P450scc. 3P-HSD. and

P450c 17 (Xiong and Hales, 1994; Bosmann et al.. 1996) and the decline in StAR

production (Bosmann et al., 1996). It is conceivable that LPS may act similarly in

marnmals and teleosts given the great homology between the testicular steroid

biosyntlietic pathways of both classes. Also, it was demonstrated in the present study that

LPS exerted effects on T levels in both the in vitro and in vivo experiments, therefore, the

activity of LPS is not dependent on the involvement of the H-P-1 avis or pituitary

gonadotropins.

Testosterone levels in goldfish with testes in an advanced stage of the

spermatogenetic cycle, as determined by gross morphology and GSI value (GSD1 .O),

were not reduced in response to LPS compared with goldfish with Iower GSI values

( G S I 4 .O). Although considerable work is needed to substantiate this, an interesting

hypothesis c m be projected from these results. Studies by Loir et al. (1995) have

demonstrated that testicular macrophages in trout are most numerous in testes that are

either regressed or are resuming spermatogenesis. It may be hypothesized that LPS

injection into goldfish in the CSI4.O treatment group resulted in the activation of

testicular macrophages and subsequent secretion of cytokines which, in tum, exened

inhibitory effects on testosterone production. Another explanation for LPS not affecting

T levels in goldfish in the GSI> 1 .O treatment group would be that the considerable

variation in plasma T levels between fish in this group obscured any treatment effect.

There are few reports of the effects of LPS in mmmals in vitro, and al1 studies

have been conducted on ovanan steroidogenesis. However. the inhibitory effect of LPS

on gonadotropin-stimulated T levels observed in the present study is similar to the

inhibition of LH-stimulated estradiol accumulation from rat granulosa ce11 cultures

(Taylor and Terranova, 1996). There are no reported effects of LPS on basal gonadal

steroid production in vitro but the potentiated T levels in my study may be due to LPS-

induced stimulatory factors. Prostaglandins, namely PGEZ, may have mediated the LPS

effect as they have been s h o w previously to stimulate T production by goldfish testis

pieces (Wade and Van Der Kraak, 1993; Wade and Van Der Kraak, 1994) and are

inducible by LPS and cytokines in fish (Rowley et al., 1995). In addition to the possible

indirect effects of LPS on T production in goldfish, direct effects should be considered

also. Although there is no evidence for LPS to affect testicular steroidgenic cells directly,

human granulosa cells have been found to bind LPS (Sancho-Tella et aï., 1992) and

Taylor and Terranova (1 996) postulated that LPS directly reduced estradiol secretion by

68

rat granulosa cells.

Western blots indicated that trout macrophages of the RTS 1 1 ce11 line respond to

LPS by upregulating and secreting cytokine-like peptides antigenically similar to mouse

TNFa ( 17 kDa) and hurnan IL- 1 j3 (1 7 D a ) . IL- 1 -1ike peptides of mitogen-stimulated

carp (Verburg-van Kemenade et al., 1995) and catfish (Ellsaesser and Clem, 1994)

leukocytes have been identified by Westerns and revealed high (70 m a ) and low (15,22

kDa) molecular weight species. Trout (Zou et al.. 1999b) and carp (Fujiki et al., 2000)

IL- 1 P gene has 49-57% amino acid sequence similarity to mammalian IL- 1 P and 5 1%

similarity to ,Yenopus laevir IL- 1 B (Zou et al., 2000). The fish IL- 1 j3 precursor molecule

has a predicted molecular weight of 29 kDa and although the teleost sequence lacks an

interleukin-1 converting enzyme (ICE) cut site, a mature peptide of 18.7 kDa is predicted

(Zou et al., 1999b). The high and low molecular weight species visualized on the

representative blot in my study may be foms of the IL-@ precursor and mature peptides,

although the enzyme responsible for the cleavage of the precursor to the mature peptide is

unknown in fish.

There are fewer reports of TNF compared with IL- 1 in fish and no reports

containing Westem blot analyses of fish TNF-like proteins. In the present study, the

molecular weights of the visualized ï?W-like peptides are sirnilar to the secreted TNFa

protein in humans (1 7.3 kDa) (Sprang and Eck, 1992). Recently the TNF gene in brook

trout (Bobe and Goetz, in press) and in iapanese flounder (Hirono et al., 2000) was

sequenced. Although the flounder TNF amino acid sequence is 29% and 3 1% similar to

human RIFa and human lymphotoxin a, respectively, Hirono et al. (2000) believe the

teleost TNF gene more closely resembles mammalian TNFa due to its inducible

expression by mitogens and its genetic structure. In the present study, TNF-like proteins

were detected only in supernatants of LPS-stimulated RTS 1 1 cells which is consistent

with the study by Hirono et al. (2000) that demonstrated the upregulation of the flounder

TNF gene by LPS.

Although Bobe and Goetz (2001) detected TNF mRNA in the testis of trout,

flounder TNF gene expression was not detected in the gonads (Hirono et al., 2000). In the

present study, Western blots of testis and kidney homogenates did not detect TNF-like or

IL- 1-like proteins in control and LPS-injected goldfish, nor was TNF detected in

homogenates immunoprecipitated with an anti-mouse TNFa antibody. The same primary

antibodies used in the Western blots of RTS11 supernatants were employed in the

Westerns of tissue homogenates. The absence of immunoreactive bands in the

homogenates may be due to the absence or very low quantities of the cytokines in the

tissues or insufficient binding of the mammalian antibodies to the putative fish cytokines.

It is possible that the level of induction was not sufficient to allow their detection with the

Western procedure or that the levels of the cytokines peaked earlier on in the course of the

study not allowing their detection afier 3 days of LPS exposure.

In summary, this is the fint study to dernonstrate that bacterial endotoxin

modulates goldfish testosterone production in vivo and in vitro. As well, it was

demonstrated that RTS 1 I ceils produce cytokine-like peptides that are antigenically

similar to mouse TiWa and IL-1 P. The cytokines were only detectable in Western blots

of LPS-stimuiated RTS 1 1 ce11 supernatants. However, Western blots did not reveal TNF-

or IL-like proteins in kidney and testis homogenates. These results, combined with the

results of Chapter 2, provide support for the hypothesis that immune activation in teleosts

alters testicular steroidogenesis and that this modulation of steroid production may be

mediated by cytokine activities. Yet, further investigations are required to discern if these

cytokines are produced in measurable quantities within the testis.

CHAPTER 4

General Discussion

The results of the present study are consistent with the belief that immune-

endocrine signalling developed early in evolution (Balm, 1997) and that this bidirectional

communication may govem multiple processes, including reproduction. The current

concept of concerted actions between endocrine and immune systems was originally

described in relation to mammals (Blalock, l989), although evidence that these systems

are integrated during conditions of stress in fish has been available for several years

(reviewed in Balm. 1997). This bidirectional communication exists to ensure that

challenges perceived by one system are translated into an appropriately integrated

response. Communication between the immune and endocrine systerns is facilitated by

the presence of cornmon messengen (cytokines and hormones) and receptors. Although

receptors for hormones (e.g. corticosteroids) have been demonstrated in leukocytes of

fish (Balm, 1997), there are no reports of the presence of receptors for TNF and IL- 1 P (or

any cytokine) on endocrine cells. However, the activity of mammalian recombinant

TNFa and IL- 1 P in the present study implies the presence of homologous receptors in

goldfish testis. Future studies could employ antibodies to mammalian cytokine receptors

in the in vitro incubations used in the present study to determine the presence of

homologous receptors in testis. Such an approach has been taken in Iang et al. (1995a),

in which antibodies to marnmalian RIF-RI were used to ablate the responsiveness of

teleost phagocytes to human TNFa implying the presence of a fish TNF receptor.

In mammals, the effects of TNFa and IL4 overlap to a high degree (Chaplin and

Hogquist, 1992). As inhibitory effects of LPS and rnurine recombinant TNFa and IL-1 j3

on T production by goldfish testis were observed in the present study, the same may hold

(rue for the teleost equivalents of IL-1 and TNF. These are similar effects to those

described in assays using marnmalian Leydig ce11 preparations (reviewed in Hales, 2000).

Likewise, the mechanism by which TNFa modulated gonadotropin-stimulated T

production is similar to marnmalian reports in that its major site of action is post-CAMP

generation but prior to the conversion of pregnenolone to other sterol precursors of T. To C

a lesser extent, the enzyme 3P-HSD may be affected by TNFa and future studies

concerning the mechanism of TNFa in teleosts should consider the possibility of multiple

lesions along the steroid biosynthetic pathway.

Studying the effects of LPS on endocrine tissues represents an experimental

approach that does not require homologous factors, but which strongly substantiates the

operation of immune-endocrine signalling in vertebrates (Balm, 1997). As in mammals,

LPS preparations have been known for several years to affect the H-P-I axis in vivo in

fish, generally in a stimulatory manner (White and Fletcher, 1985; Balm et al., 1995).

The results of my study suggest that the communication between the immune and

endocrine systems in teleosts is not limited to within the H-P-I axis. but may affect the H-

P-G axis and govem reproductive processes. This was demonstrated through the

administration of LPS to goldfish testis pieces in vitro and to male goldfish in vivo which

resulted in altered Levels of testosterone. The mechanism by which LPS affects gonadal

steroidogenesis in teleosts is unknown but it is reasonable to hypothesize that the

modulation of steroid production was due to an increase in cytokine production following

LPS treatment in both the in vivo and in vitro experiments given the data provided by

similar mammalian studies (O'Bryan et al., 2000a). While the present study provides the

first evidence of cytokines exerting effects on teleost gonadal tissue, other endocrine

tissues in fish have been shown to be affected by recombinant mammalian IL-l and

TNFa, as well as LPS (reviewed by Balm, 1997). It is possible that in the in vivo

experiments, testosterone levels were altered through the activation of the H-P-1 avis

(Pankhurst and Van Der Kraak, 1997) and/or LPS-induced cytokines may have acted at

the level of the gonad.

The physiological relevance of cytokine-mediated inhibition of testosterone

production in fish has not been addressed yet, but a hypothesis based on mammalian

studies has been proposed by researchers in this field of study (Hales. 1997). In most

mammalian species studies, males have weaker immune responses than females. The

weaker immune response contributes to higher susceptibility to infection, and poorer

sumival of males compared to females. Studies in many experimental models have

established that the underlying basis for this sex-specific difference in susceptibility is

due to differences in gonadal steroids androgens which are known to be

immunosuppressive. These observations, together with the results of studies that indicate

that cytokines are elevated during conditions associated with decreased testosterone,

provide the basis for the hypothesis that for an animal to wage the maximum possible

immune response, immune components (cytokines) are required to inhibit the production

of immunosuppressive androgens (Hales, 2000). While the present study indicates that

pro-inflammatory cytokines and LPS inhibit testicular T production in goldfish, it is

necessary to take into consideration previous studies that indicate that fish are more

tolerant to LPS than higher vertebrates (e.g. White and Fletcher, 1985). The low

sensitivity of fish to LPS may be related to natural defense mechanisms and may be one

of the ways in which fish adapt to their environment (Kodama et al., 1987). Therefore, it

may be postulated that mechanisms which regulate the production of cytokines in fish

will di ffer or exhibit altered levels of sensitivities compared with marnrnals.

It was shown in the present study that the RTS 1 I macrophage ce11 line responds to

LPS by upregulating the production of TNF- and IL- I-like peptides as detected by

Westem blots using antibodies to mammalian TNFa and IL- 1 P. The visualized bands are

similar to the known molecular weights of mammalian TNFa. as well as to the predicted

molecular weight of fish IL- 1 P precursor and mature peptides which are quite sirnilar in

size to mammalian IL- 1 P peptides (DinarelIo, 1997). There are no previous reports

indicating that RTS 1 1 secretes TNF- and IL-1-like peptides, and there are no published

studies demonstrating the presence of TNF-like peptides in fish via Westem blotting

techniques. However, the same mammalian antibodies that cross-reacted with factors in

the RTS 1 1 supematants did not detect the presence of these factors in the testes or

kidneys of male goldfish. Funher work is required to determine the potential role of

these cytokines as paracnne regulaton of testicular function in fish and key to these

investigations will be the detection of secreted cytokines within the testis.

Investigations into the intratesticular production of these cytokines may require

the use of more sensitive and specific techniques such as the use of antibodies direcied

against fish TNF and I L 4 P. The recent sequencing of teleost I L 4 P and TNF genes now

makes it possible to determine their potential expression in gonadal tissues by molecular

techniques (e.g. RT-PCR) or their cellular source (e.g. in situ PCR). It should not be

assumed that teleost testicular macrophages are a source of proinfiammatory cytokines

simply because the RTS 1 1 macrophage ce11 line, which was generated frorn rainbow trout

spleen, produces cytokine-like molecules. A considerable amount of work is required to

further characterize teleost testicular macrophages and pioneenng work is needed to

identify their secretory products and other potential sources of gonadal cytokines (e.g.

endocrine cells. infiltrated immune cells).

The results of the present study warrant further investigations into the activities of

cytokines and other macrophage-derived factors in teleost reproductive processes. At

present, a major obstacle in evaluating the effects of cytokines on teleost testicular

functioning is the lack of fish recombinant TNF and IL- 1 P and the assay systems to detect

them in lower-vertebrates. Future studies will need to incorporate recombinant fish

cytokines and antibodies directed against fish cytokines into expenments, such as those

found in this study, to verib results that have been found using mammalian cornpounds.

Characterization and purification of the active factor(s) in fish leukocyte supernatants,

and cloning the genes that encode them, will allow more extensive investigations into

their potential replation of reproductive processes and mechanisms of action. In

addition to studying how cytokines may regulate reproductive events, deciphering the

cross-talk that most likely occun between signals of both the immune and endocrine

systems in teleosts will further the understanding of how fish regulate reproductive

processes.

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