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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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