the b rain -pitu itary-gonadal axis of the three -spined ...555528/fulltext02.pdf · the brain...
TRANSCRIPT
The brain-pituitary-gonadal axis of the three-spined
stickleback, Gasterosteus aculeatus
Yi Ta Shao
Department of Zoology
Stockholm University
2012
The brain-pituitary-gonadal axis of the three-spined stickleback, Gasterosteus aculeatus
Doctoral dissertation 2012
Yi Ta Shao [email protected] ; [email protected] Department of Zoology Stockholm Universirty SE-106 91 Stockholm Sweden
© Yi Ta Shao, Stockholm 2012. ISBN 978-91-7447-568-5 Cover illustration by Bertil Borg Printed in Sweden by US-AB, Stockholm 2012 Distributor: Department of Zoology, Stockholm University
To
My family
0
The brain-pituitary-gonadal axis of the three-spined
stickleback, Gasterosteus aculeatus
Doctoral dissertation 2012 Yi Ta Shao Department of Zoology Stockholm University 106 91 Stockholm
Abstract
The seasonal reproduction of the three-spined stickleback is stimulated by long day photoperiod. As in other vertebrates, the reproductive system of stickleback is regulated by the brain-pituitary-gonadal (BPG) axis which is largely controlled by feedback effects. Both negative and positive feedback effects on the BPG axis have been found in fish. So far, the roles feedback effects on the BPG axis play in the photoperiodic regulation of seasonal reproduction are still unclear. This thesis focused on the photoperiodic regulation and gonadal feedback effects on the gene expressions of gonadotropin (GtH) and gonadotropin-releasing hormones (GnRH) in the brain and pituitary, and how gonadal feedback regulated the steroid homeostasis in stickleback. Both GnRH2 and GnRH3 mRNA was found in the hypothalamus. Higher expression levels of both GnRH2 and 3 in breeding than in post-breeding males suggested that they are both involved in seasonal reproduction. There was no evidence for a role of GnRH3, which may be the dominating form, in the photoperiodic control of reproduction. However, the polarity of the feedback effect on gnrh3 gene expression may turn from positive to be negative when the males went into post-breeding state. Tapeworm, Schistocephalus solidus, infection inhibited the reproduction of sticklebacks. However, the infection caused higher expression levels of both GnRHs and GtHs genes, which may be due to feedback effect on the BPG axis.
Under short day, both lh-β and fsh-β were suppressed by low androgen levels. This negative feedback may inhibit maturation completely, unless a rise of androgens triggers positive feedback under long day. The change in feedback polarity may result in all or nothing maturation. Furthermore, the androgen inhibitory effect on lh-β and fsh-β under short day could be abolished by aromatase inhibitor, which means the estrogen may cause negative feedback in males under short day.
There was no compensation effect on plasma androgen level in fully mature hemi-castrated fish. However, both testosterone and 11-ketoandrostenedione treatments increased plasma levels much less in sham-operated fish than in castrated ones, indicating that homeostatic mechanisms are nevertheless present.
1
TABLE OF CONTENTS
LIST OF PAPERS 2
INTRODUCTION 3
Reproductive rhythms of fish 3
Brain-pituitary-gonadal axis 4
Gonadotropins (GtH) 5
Gonadotropin-releasing hormones (GnRH) 6
Reproduction of the stickleback 9
Infection with the parasite, Schistocephalus solidus 11
AIM OF THE THESIS 12
MATERIAL AND METHODS 13
Experiment animal 13
Treatments and sampling 13
Implants 13
Operation 14
Sampling 14
Experimental protocol 15
Cloning and bioinformatic analysis 18
Real time quantitative PCR (q-PCR) 19
GnRH 19
GtH 20
In situ hybridization 20
Radioimmunoassay (RIA) 22
Statistics 22
RESULTS AND DISCUSSION 22
GnRH gene expression in stickleback (paper I, II) 22
The feedback effect on GtH gene expression (paper III) 25
Homeostasis of circulating androgens levels (paper IV) 27
SUMMARY 28
ACKNOWLEDGEMENTS 30
REFERENCES 31
2
LIST OF PAPERS
Paper I Yi Ta Shao, Yung Che Tseng, Chia Hao Chang, Pung Pung Hwang and
Bertil Borg. 2012. GnRH mRNA levels in the male three-spined
stickleback, Gasterosteus aculeatus; effects of photoperiod,
gonadectomy and breeding-postbreeding condition. MS
Paper II Yi Ta Shao, Yung Che Tseng, Susanne Trombley, Pung Pung Hwang,
Monika Schmitz and Bertil Borg. 2012. Schistocephalus solidus
infections increase gonadotropin and salmon gonadotropin releasing
hormone mRNA levels in the three-spined stickleback, Gasterosteus
aculeatus. Parasitol. Int. 61: 470-474.
Paper III Yi Ta Shao, Mia Arvidsson, Susanne Trombley, Rüdiger Schulz, Monika
Schmitz and Bertil Borg. 2012. Androgen feedback effects on LH and
FSH, and photoperiodic control of reproduction in male three-spined
sticklebacks, Gasterosteus aculeatus. MS
Paper IV Yi Ta Shao, Rüdiger Schulz and Bertil Borg. 2012. Homeostasis of
circulating androgen levels in the male three-spined stickleback
(Gasterosteus aculeatus). MS
3
INTRODUCTION
Reproductive rhythms of fish
Reproduction is often a cyclic phenomenon in fish. In many tropical fishes, the
relatively constant environment allows them to have extended spawning seasons
which may include multiple breeds throughout the year (Pauly, 1998; Petersen &
Warner, 2002). Other fishes, however, especially those living in the temperate
zones or other places with clear seasonal climatic variability, have a predictable and
restricted spawning period. The reproduction periods of these species usually last
only several weeks or even just a couple of days. The specific reproductive and
maturational timing of fishes is a result of adaption and evolution. It is critical for
the male and female to be ready to breed at the same time and to ensure the young
hatch and commence feeding in the right season (Billard and Breton, 1978).
In order to spawn at the right time, fishes use both environmental cues and
endogenous cycles for timing of growth and maturation. Various teleosts, due to
their unique evolution and adaptations, can use almost all occurring environmental
events as exogenous timing factors. Many environmental cues, such as
photoperiod, temperature, salinity, lunar phase, pH, food abundance and social
interactions, are known to regulate spawning and sex maturation, (Stacey, 1984).
Nevertheless, how these external factors regulate the occurrence of maturation and
spawning is not clear.
The photoperiod is the most well-studied factor that synchronizes seasonal
reproduction timing. It is the major cue used in many temperate species such as
salmons, sticklebacks and seabasses (Begtashi et al. 2004; Bromage et al., 1994;
Howell et al. 2003; Randall et al., 1998 Borg 2010). The earliest recorded
photoperiodic experiment on fishes was conducted in 1937 and used the brook trout
(Salvelinus fontinalis). The experiment demonstrated that when the trout were
exposed to increasing light phase early in the year followed by rapidly decreasing
light phase, their second breeding season would appear 3 months earlier than that of
the control group (Hoover & Hubbard, 1937). Changing photoperiods have been
4
observed to accelerate maturation or advance spawning time also in many other
species of Salmonidae, such as rainbow trout (Oncorhynchus mykiss, Bromage, 1992),
coho salmon (O. kisutch, Withler, et al., 1998), masu salmon (O. masou, Amano et al.,
1995) and Atlantic salmon (Salmo salar, Taranger, et al., 1998). In the three-spined
stickleback (Gasterosteus aculeatus), sexual maturation is stimulated by long
photoperiod, but inhibited by short photoperiod (Baggerman, 1985; 1989; Borg,
1982a; Borg, 2010).
The use of artificial photoperiod manipulation has increasingly become a
standard operating practice in the aquaculture industry to control maturation or
breeding of a number of species, such as Atlantic salmon (Porter et al. 1999; Endal et
al. 2000), striped trumpeter (Latris lineate) (Morehead et al. 2000) and European
seabass (Dicentrachus labrax) (Begtashi et al. 2004). It is also effective in Atlantic
cod (Gadus morhua) (Davie et al. 2003; Hansen et al. 2001; Almeida et al. 2009) and
in other species, such as gilthead seabream (Sparus aurata) and white seabass
(Atractoscion nobilis) (Aalbers, 2008; Perdikaris, 2001).
However, the mechanism of how photoperiods influence the reproductive
process of fishes is still unclear. Yet it is known that fishes synchronize their
physiological functions to the light/dark cycle, and that the endogenous periodicities
of physiological processes are responsible in part for seasonal reproduction.
Brain-pituitary-gonadal axis
As in other vertebrates, the reproductive system of fishes is regulated by the
brain-pituitary-gonadal endocrine axis which is regulated by feedback effects (Fig 1).
Both positive (stimulating) and negative (suppressing) feedback effects on the
BPG-axis have been reported in fishes (Crim and Evans, 1979, Billard et al., 1977;
1978). For example, gonadal hormones, i.e. testosterone (T), 11-ketotestosterone
(11KT) or estradiol (E2) acting on the pituitary and/or brain level, may act on the
pituitary and/or brain level to suppress or stimulate gonadotropin (GtH) secretion.
The negative feedback effects on the BPG-axis are important mechanisms to keep the
levels of sex steroids stable; and the positive feedback may play an important role in
5
controlling the onset of reproductive events in fishes.
Figure 1. A proposed model of the Brain-pituitary- gonadal axis. Feedback loops control the synthesis and concentrations of sex hormones. GtHs: gonadotropins; GnRH: gonadotropin-releasing hormone; GnIF: gonadotropin inhibitory factors; T: testosterone; E2: estradiol.
Gonadotropins (GtHs)
Teleosts have two types of gonadotropins - luteinizing hormone (LH) and
follicle-stimulating hormone (FSH) as in tetrapods. They share a similar α peptide
chain but have different β chains. The two gonadotropins stimulate different
receptors, due to the specific β chain.
The gonadotropins contents in the blood or pituitary can be measured by
radioimmunoassy (RIA). However, a specific antibody is required for this method,
and it is unpractical to be made in a small species. On the other hand, the synthesis
rate of GtHs can be estimated by measuring the lh-β or fsh-β mRNA content of the
pituitary, which makes it possible to study the activity of GtH in relative small species,
such as three-spined stickleback (e.g. Hellqvist, et al., 2006).
Gonadotropin-releasing hormones (GnRH)
The secretion of GtHs is controlled by the hypothalamus, which can be
stimulated by gonadotropin-releasing hormone (GnRH) (Goos, 1991; Peter, et al.,
6
1991), or inhibited by other substances, such as the peptide -
gonadotropin-inhibitory hormone (GnIH) (Tsutsui et al. 2012) and dopamine
(Goos, 1991; Peter, et al., 1991). The primary structure of GnRHs contains an
amino-terminus (pGlu), a NH2 terminus and in all 10 amino acids. The first
GnRH isoform characterized was isolated from mammalian hypothalamii and
was named mammalian GnRH (mGnRH). mGnRH is known as a hormone that
regulates the reproductive system by stimulating release of gonadotropic
hormones from the anterior pituitary gland (Burgus et al., 1972). mGnRH is the
most widespread form in mammals, and is also found in European eel (Anguilla
anguilla) (King et al., 1990). In birds, two GnRH molecular variants have been
isolated from chicken brains, cGnRH-I (King and Millar, 1982) and cGnRH-II
(Miyamoto et al., 1984). cGnRH-II is not only present in birds, but also in many
fishes such as European eel (King et al., 1990), African catfish (Clarias gariepinus)
(Goos, 1989) and goldfish (Carassius auratus) (Yu et al., 1988). The first form
characterized from fish is sGnRH which was isolated from chum salmon
(Oncorhynchus keta) by Sherwood et al. (1983).
At present, more than 11 structural variants of GnRH have been isolated
and characterized from fishes and lamprey (Table 1). Almost all fish species
investigated have at least two GnRH forms in the central nervous system; and
most bony fishes have three forms of GnRH (review in Somoza, et al., 2002).
Fernald and White (1999) proposed that the multiple GnRH forms evolved from
3 different evolutionary lineages; the mesencephalic, telencephlic and
hypothalamic lineages.
According to the features of the peptide sequences and the dispersal
pattern of GnRH neurons, those isoforms can be divided into different groups. In
teleost, they are GnRH1, GnRH2 (cGnRH II) and GnRH3 (sGnRH) (review in Okubo
& Nagahama 2008). GnRH2 has been found in all studied teleost species, both in
Chondrichthyes and Osteichthyes (Somoza et al., 2002). GnRH3 is also
widespread amoung fishes, but is absent in Chondrichthyes, sturgeons,
lungfishes and in some teleost species, i.e. African sharptooth catfish (Clarias
gariepinus) and broadhead catfish (Clarias macrocephalus) (Bogerd et al., 1994;
7
Ngamvongchon et al., 1992). The gene of hypothalamic GnRH (GnRH1) lineage
can be missing in some species, such as goldfish (Carassius auratus) (Lin and
Peter, 1996 [22]) and masou salmon (Oncorhynchus masou) (Suzuki et al., 1992
[23]). Moreover, almost the whole genome of zebrafish (Danio rerio) has been
sequenced, but the gene of GnRH1 has not been found in this species (Steven et
al. 2003; Palevitch et al. 2007). A possible reason is that the gene of this GnRH
lineage has been lost or become a pseudogene during evolution [review in
Okubo & Nagahama 2008]. In the species which do not have hypothalamic
GnRH, such as salmon, zebrafish or goldfish, telencephalic GnRH (GnRH3) may
take on the role [review in Okubo & Nagahama 2008]. The GnRH3 expressing
neuron were seen in the pre-optic area, as well as in the terminal nerve ganglion,
which project their axons into the pituitary, indicating that sGnRH would act as
the main stimulator of the pituitary hormones in the absence of hypothalamic
GnRH (Okubo and Nagahama, 2008).
Table 1. The GnRHs found in fish, included lamprey (Partly summarized from Somoza, et al.
2002)
GnRH form Species Authors sGnRH chum salmon Sherwood et al., 1983 (Oncorhynchus keta) lGnRH I sea lamprey Sherwood et al., 1986 (Petromyzon marinus) lGnRH II sea lamprey Sower et al., 1993 cfGnRH broadhead catfish Ngamvongchon et al., 1992 (Clarias macrocephalus) dfGnRH spiny dogfish Lovejoy et al., 1992 (Squalus acanthias) snGnRH snook Sherwood et al., 1993 (Centropomus undecimalis) sbGnRH seabream Powell et al., 1994 (Sparus aurata) hrGnRH herring Carolsfeld et al., 2000 (Clupea harengus pallasi) wfGnRH whitefish Adams et al., 2002 (Coregonus clupeaformis) mdGnRH medaka Okubo et al., 2000 (Oryzias latipes) pjGnRH pejerrey Montaner et al., 2001 (Odontesthes bonariensis) ?GnRH Tilipa Cao & Ding, 2006 (Oreochromis aurea) The GnRHs listed below are present in fishes,
but were first identified from other species mGnRH rat Burgus et al., 1972
8
(Rattus norvegicus) cGnRH I chicken King and Millar, 1982 (Gallus gallus) cGnRH II chicken Miyamoto et al., 1984
sGnRH is also widespread among fishes, but is absent in Chondrichthyes,
sturgeons, lungfishes and in some teleost species, i.e. African sharptooth catfish
(Clarias gariepinus) and broadhead catfish (Clarias macrocephalus) (Bogerd et al.,
1994; Ngamvongchon et al., 1992) (Fig 2).
Figure 2. Hypothetical scheme for the evolution of gonadotropin-releasing hormone (GnRH) in vertebrates. hypothalamic lineage (GnRH 1), mesencephalic lineage (GnRH 2) and telencephlic lineage (GnRH 3). Mya: million years ago. (Okubo and Nagahama, 2008)
In teleost, the GnRH1 vary between different taxa, the seabream form
(sbGnRH) is limited to some orders in Acanthopterygii (review in Somoza et al.,
2002), and the catfish form (cfGnRH) has only been found in the genus Clarias
(Ngamvongchon et al., 1992; Bogerd et al., 1994). However, the gene of
hypothalamic GnRH lineage may be missing in some species, like goldfish
(Carassius auratus) (Lin and Peter, 1996) and masou salmon (Oncorhynchus
masou) (Suzuki et al., 1992) (Fig 3).
rhesus
9
Figure 3. Schematic diagram illustrating the molecular evolution of hypothalamic lineage GnRH in vertebrates. Its ancestral form was certainly mGnRH. It underwent great structural diversification in non-mammalian lineages, leading to 6, 2, 1 and 1 structural variant in the teleost, amphibian, reptilian and avian lineages respectively. Representative species having the respective molecular forms are shown. (Okubo and Nagahama, 2008)
Reproduction of the stickleback
The three-spined stickleback, Gasterosteus aculeatus, is one of the most
studied fishes as regards the control of seasonal reproductive cycles (Borg, 2010).
The natural breeding season of the species is late spring-early summer when
photoperiods are long and temperature high. Breeding male sticklebacks have bright
breeding colours, with blue eyes and red belly, and show nest building behavior. The
nest is built of algae glued together with threads of the protein spiggin produced by
the kidney, which hypertrophies under androgen stimulation (Jakobsson et al. 1999;
Borg, 2007).
After the breeding season, these characters disappear. In the yearly cycle, the
highest kidney development is found in the breeding season and the lowest in the
post-breeding period in late summer (Borg, 1982). A similar pattern is also found in
plasma levels of the androgen 11-ketotestosterone (Mayer et al., 1990) and in
pituitary lh-β and fsh-β mRNA levels (Hellqvist et al., 2006). Long photoperiod, but
not short photoperiod, stimulates maturation in the stickleback (Baggerman, 1957;
Borg, 1982). Both lh-β and fsh-β mRNA level are higher in males kept under long
photoperiod (L:D 16:8) than under short photoperiod (L:D 8:16) in winter (Hellqvist,
et al. 2008).
Andersson et al. (1995) found three distinct groups of GnRH-ir perikarya in the
10
stickleback brain; in the nucleus olfactoretinalis, the nucleus anterioris
periventricularis and in nucleus lateralis tuberis. The proximal pars distalis of the
pituitary, where the gonadotropic cells are located, was innervated by GnRH-ir fibers.
Via high performance liquid chromatography (HPLC) combined with
radioimmunoassay (RIA), at least two peaks with sGnRH-immunoreactive material
using of whole stickleback brain extracts has been identified. One form was
tentatively identified as sGnRH (GnRH3), whereas the other(s) are still unclear
(Andersson et al., 1995). In genome databases (Ensembl Genome Browser system),
two types of GnRHs, GnRH2 (cGnRH II) and GnRH3 (sGnRH) have been found in DNA
level (Fig 4a, b). However, GnRH1 is absent in stickleback genome. Even though the
neighbor genes of GnRH1 were found on chromosome XII, no possible GnRH gene
was present in the appropriate area (Fig 4c) (Tostivint 2011).
a.
b.
11
Figure 5. The parasite S. solidus and its host. The numbers indicate the totally 8 parasites found in this stickleback (photo by Yi Ta Shao)
c.
Figure 4. Synteny map comparing the orthologues of the (a) GnRH2, (b) GnRH3 and (c) GnRH3 locus and the genes flanking it in humans (Homo sapiens), chicken (Gallus gallus), Clawed frog (Xenopus tropicalis), medaka (Oryzias latipes), stickleback (Gasterosteus aculeatus), pufferfish (Tetraodon nigroviridis) and zebrafish (Danio rerio). (Tostivint 2011).
Infection with the parasite, Schistocephalus solidus
The three-spined stickleback is an intermediate host for the cestode S. solidus
(Fig 5). The life cycle of S. solidus includes a free swimming larval stage (coracidium),
a second larval stage hosted in cyclopoid copepods (procercoid), a third larval stage
hosted in the stickleback (plerocercoid) and an adult stage in birds (Fig 6). When the
fish is eaten by a bird, the plerocercoid will mature quickly and start to reproduce
within a few weeks (Schultz et al. 2006). The parasitized fish often has a distended
abdomen which contains one or multiple worms. The total mass of those parasites
may be close to, or even exceeding, the
mass of their host fish. S. solidus can
have a devastating effect on stickleback
sexual maturation in some populations,
but less in others (McPhail and Peacock,
1983). In the cases of Scout Lake (Heins
and Baker, 2003) and Walby Lake, Alaska
(Heins and Baker, 2003; Heins, et al.,
2010), most of the host females
matured eventually, despite the eggs
mass of those females may be lower than in uninfected fish. The infected males
2 1
3 4
5
8
7
6
12
collected from Victoria Park pond had lower kidney somatic index (KSI) and were less
aggressive in breeding behavior than uninfected males. However, no difference in KSI
or reproductive behavior was found between the infected and uninfected males from
Kendoon Loch (MacNab et al., 2009),). Sexual maturation of infected sticklebacks
may be related to the mass of parasites, with more heavily infected fish being less
like to mature (review in Barber, 2007), or due to the genotypes of the host
populations (MacNab, et al. 2009).
Figure 6. Life cycle of S. soldus. a. free swimming larvae, b. 2nd stage larvae host in copepods, c. 3rd stage larvae in stickleback, d.final host in birds. (Dubinina, 1980)
AIM OF THE THESIS
The main objective of this thesis is to understand the BPG axis response to the
photoperiodic seasonal reproduction by using three-spined stickleback as the model
animal. This thesis focused on the photoperiodic regulation and gonadal feedback
effects on the genes expressions of GnRH and GtH in the levels of brain and pituitary.
This study aimed to find out:
Which GnRH forms are present in the three-spined stickleback? What role do
they play in the control of seasonal reproduction and in gonadal feedback?
How does Schistocephalus solidus influence the host’s GnRH and GtH genes
expression?
Does photoperiod change the feedback effects on lh-β and fsh-β and cause
all-or-nothing maturation?
Are androgen levels in male stickleback regulated by homeostasis?
a. b.
c. d.
13
MATERIAL AND METHODS
Experimental animals
Except for paper II, the sticklebacks were caught in the Ö resund, southern Sweden
during the winter and transported to Stockholm University. The animals were stored
in aquaria containing artificial brackish water (0.5% salinity) which was aerated and
filtered. The bottom was covered with sand; ceramic pots and tubes provided hiding
places. The fish were feed daily with frozen bloodworms, Artemia or mysids.
Treatments and sampling
Implants
Eight types of implants were used in paper III and IV to carry drugs into the
fish body (Table 2). All implants were made from medical grade Silastic tube (ID
0.64mm; OD 1.19mm) which was cut to 5mm in length and filled with crystalline
T (Fluka), crystalline 11-ketoandrostenedione (11KA) (4- Androstene- 3,11,17-
trione; Sigma) or aromatase inhibitor (AI, fadrozole; CGS 16949 A, 4-(5,6,7,8-
tetrahydroimidazo[1,5-α]pyridin-5-yl) benzonitrile; a gift from Novartis). Both
ends were sealed with silicone glue. For high dosage of 11KA (11KA(H)), the
ordinary (low dose) 11KA implants (11KA(L)) were perforated three times with a
31G syringe needle. 11KA is a non-aromatisable androgen, whereas it can be
converted to 11-ketotestosterone (11KT) in the fish.
Other types of T implants were made from 5 mm Silastic tubes, but filled
with different concentrations of T (0.10; 0.20; 0.25 or 0.50 %) dissolved in cacao
butter, and not sealed at the ends. Those implants made it possible to give a
lower dosage than by using crystalline implants. The cacao butter-implants were
stored at 4 ⁰C before use. Empty implants or the implants filled with cacao butter
alone were used as controls.
The experiments were carried out with the permission of the Stockholm
Northern Animal Experiment Ethical Committee
14
Operations
Fish were either castrated or sham-operated and were given different types
of implants. The fish were anaesthesized with c. 0.1% 2-phenoxyethanol (up to
2009) or 0.025% MS-222 (Ethyl 3-aminobenzoate, methanesulfonic acid salt)
solution (0.25 g MS-222 buffered with 0.50 g sodium bicarbonate dissolved in
1.0L water) (from 2010), c. 1.5 mm long incisions were made into the abdominal
cavity on each side and the testes were excised with fine forceps. Sham-operated
fish were treated similarly, but the testes were not removed. The incisions were
closed with BV-2 (0.4 Ph. Eur) suture. In some categories of fish were marked
with spine-clipping. After the operation, the fish were put into aquaria and kept
under different photoperiods.
Sampling
All fish were euthanized with 0.1% 2-phenoxyethanol (up to 2009), or with
0.025% buffered MS-222 solution (from 2010) before dissection. The body and
kidney were weighed, and the breeding color and the kidney maturity status
were recorded. Kidney-somatic index (KSI, kidney weight / (body weight-
parasite weight)) or gonadal-somatic index (GSI, ovary weight/(body weight-
parasite weight)) and parasitation index (PI, total parasite weight/(body weight-
parasite weight)).
The brain and pituitary were excised separately after decapitation and
removal of the skullcap. Whole brains with 2-3 mm of myelencephalon were
used, including the olfactory lobes, which is one of the areas where
GnRH-immunoreactive perikarya are distributed in the stickleback (Andersson et
al. 1995). The pituitary was separated from the brain after decapitation and
removal of the skullcap, and both of them were then immersed in RNAlater®
(Ambion) (50μl for pituitary; 350μl for brain) respectively kept in 4 ⁰C for 8 hour,
before storage at -70 ⁰C until analysis.
15
The blood was collected from the caudal peduncle with micro haematocrit
tubes (Na-hep. Cat No. 7493 11; BRAND). After centrifugation for 2 min, the
plasma samples were removed to pre-weighted Eppendorf tubes. The plasma
volume was measured by weighing and plasma was stored at -70 ⁰C.
Experimental protocols
Paper I
Three experiments were done in paper I (Table 4). Moreover, there is one
additional post-breeding castration experiment only presented in the thesis
summary. Male sticklebacks were kept under long (L16:D8) or short (L8:D16)
photoperiod for 4 weeks and dissected in Feb 2009. In Nov 2009 – Jan 2010,
male fish were kept under long photoperiod to reach breeding state (with bright
breeding colour and nest), or an even longer time to post-breeding state (no
more breeding color). Gonadectomy experiments were carried out in 2010 Mar
(breeding fish) and 2011 Oct (post-breeding fish), which were dissected two
weeks after operation (breeding fish) or after 10 weeks when those males lost all
breeding colour (post-breeding fish). gnrh2 (chicken II GnRH) and gnrh3 (salmon
GnRH)) mRNA levels were measured.
Table 4. Protocols of paper I.
Group (n) LD
Long photoperiod (20) 16:8 Short photoperiod (20) 8:16 Breeding (18) 16:8 Post-breeding (17) 16:8 Castrated breeding (10) 16:8 Sham-operated breeding (10) 16:8 <The experiment below was not included in paper I> Castrated post-breeding (7) 24:0 Sham-operated post-breeding (8) 24:0
16
Paper II
Male and female three-spined sticklebacks were collected by drop nets at
the Askö Laboratory in northwestern Baltic Proper (58°49′N, 17°38′E). The
samplings were done on April 28 2010, a few weeks before the onset of the
breeding season. The fish were anaesthesized and dissected after capture. The
sample sizes of each groups are shown in Table 5. The gnrh2 and gnrh3 gene
expression levels in the brain and lh-β and fsh-β levels in the pituitary were
measured.
Table 5. Fish in paper II.
PI (%) KSI (%) GSI (%)
Males With parasite (n=8) 22.02 ± 3.59 1.16 ± 0.12 – Without parasite (n=6) – 1.88 ± 0.23 – Female With parasite (n=10) 24.38 ± 1.37 – 3.85 ± 0.42 Without parasite (n=9) – – 6.43 ± 0.56 Parasite index, kidney somatic index and Gonadosomatic index calculated as organ or total parasite weight x (body weight without parasite)-1 x 100 *p<0.05
Paper III
Four sets of experiments were done during 2007 to 2011. In general, males
were either castrated or sham-operated and treated with different implants.
After operations fish were transferred to different aquaria under different
photoperiods (Table 6). The fish were dissected approx. 1 month after operation.
The mRNA levels of lh-β and fsh-β in the pituitary were then measured. In the
experiment 2011, the plasma samples of each group were collected for T levels
measurement.
*
*
17
Table 6. Protocols of paper III. 2007 (35) 12:12 Control# Control 2009 (16) 8:16 Sham Control (10) Castration Control -10 (10) Castration Control (20) Castration T 0.25 (10) Castration T 0.25 (35) 13:11 Control# Control (10) Castration T cryst (10) Castration Control (10) Castration AI (20) Castration T 0.25 (10) Castration T 0.25 (35) 14:10 Control# Control + AI (10) Castration Control (10) Castration T cryst (20) Castration T 0.25 + AI 2008 (10) 16:8 Sham Control 2011 (16) 16:8 Sham Control (10) Castration Control (16) Castration Control (10) Castration T 0.25 (15) Castration T 0.10 (10) Castration T cryst (15) Castration T 0.25 (10) Castration 11KA (15) Castration T 0.50 (10) 8:16 Sham Control (16) 8:16 Sham Control (10) Castration Control (16) Castration Control (10) Castration T 0.25 (15) Castration T 0.10 (10) Castration T cryst (15) Castration T 0.25 (10) Castration 11KA (15) Castration T 0.50 Sample sizes (n), Sham-operated (Sham), Implants: Control (empty or cacaobutter alone), T cryst (Crystalline T), T 0.10/ 0.25/ 0.50 (0.10%/0.25%/0.50% T in cacaobutter), 11KA (Crystalline 11KA). #Controls includes both unoperated and sham-operated fish.
Paper IV
Breeding fish were either completely castrated, hemi-castrated, or
sham-operated and treated with different types of implants. Several sets of
experiments, each using 10-25 individuals were carried out in 2008, and 2009
spring (11KA and AI treatments) and in 2011 (T and AI treatments). In total, 206
fish were used. The numbers in each treatment are shown in Table 7. Plasma
samples were collected, and the levels of 11KT and T were measured via
Radioimmunoassay (RIA).
18
Table 7. Protocols of paper IV.
2008-2009 2011
Operation Implant Sample N(N)* Operation Implant Sample N(N)*
Sham Empty 29 (29) Sham Empty 13 (13) 11KA(L) 12 (12) T(1%) 10 (10) 11KA(H) 12 (12) T(2%) 11 (11) AI 12 (10) AI 10 (9) Hemi Empty 16 (16) Castr Empty 10 (0) 11KA(L) 4 (4) T(1%) 10 (0) 11KA(H) 13 (13) T(2%) 8 (6) Castr Empty 15 (0) 11KA(L) 6 (0) 11KA(H) 5 (3)
Sham operated (Sham), hemi-castrated (Hemi) or completely castrated (Castr). Empty implants or implants with 1% or 2% of testosterone (T) in cacaobutter; 11KA(H)/(L): high/low dosages of 11 ketoandrostenedione; AI: the aromatase inhibitor flutamide. *Sample number, N, and number of males with breeding colours, (N).
Cloning and bioinformatic analysis
In paper I, peptide sequences from other species (teleosts were given a highest
priority) were used to BLAST the genome databases (Ensembl Genome Browser
system) for stickleback GnRH homologs. In silico predicted full-length of stickleback
GnRH homologs obtained from the genome were carefully confirmed by the
GenBank database. Specific primers (Table. 8) were designed for the
reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. PCR products thus
obtained were subcloned into a pGEM-T Easy vector (Promega, Madison, WI, USA),
and the nucleotide sequences were determined with an ABI 377 sequencer (Applied
Biosystems, Warrington, UK). Sequence analysis was conducted with a BLASTx
program (NCBI). To verify the membership of identified candidates in the core GnRH
family, the deduced amino-acid sequences of cloned stickleback GnRHs were aligned
with ClustalX together with all known GnRH protein sequences of teleost available in
public databases.
Table 8. RT-PCR primer for cloning. Gene Primer sequence Product size(bp) gnrh2 F 5’-ATGTGTGTATCTCGGCTGGTTTTG-3’ 258 R 5’-TCACTTTCTTTTCTGAAGCTCTCT-3’ gnrh3 F 5’-ATGGAAGCGAGCAGCAGAGCGATG-3’ 273 R 5’-AAGAGAAAGTTCCAAAATAATTGA-3’
Physical gene maps of verified GnRH loci were scaled based on assemblies of the
Ensembl Genome Browser. Genes located up- and downstream of GnRH genes in
19
these loci were blasted against mammalian genomes to determine the highest score.
In addition, up- and downstream genes located in the conserved syntenies of the
mammalian GnRH were also examined in stickleback genome databases to
determine the precision of in silico cloning.
Real time quantitative PCR (q-PCR)
GnRHs
Two types of GnRHs; chicken GnRH II (cGnRH II, a GnRH 2) and salmon
GnRH (sGnRH, a GnRH 3) had been found in the stickleback genome when GnRH
sequences from other species were used to BLAST the genome databases
(Ensembl Genome Browser system) for Stickleback GnRH homologs. No other
previously known vertebrate GnRH was found and though the neighbor genes of
GnRH 1 were found on chromosome 12, but no GnRH was present in the
appropriate area, as also not found by Tostivint (2011). By means of
reverse-transcriptase polymerase chain reaction (RT-PCR), the complete cDNA
sequences of about 255~270 bp of the stickleback GnRH coding regions were
confirmed, and used to design the primers for ribosomal protein (RPL8), GnRH 2
and GnRH 3 (Table 3) specifically for three-spined stickleback.
Total RNA was extracted by TriZol reagent (Invitrogen) and
reverse-transcribed from the brains of sticklebacks according to the methods in a
previous study (Wu et al. 2009). The RNA samples were cleaned up with DNase
(2U) using TURBO DNA-free kit according to the manufacturer’s instruction
(Ambion) to remove possible genomic DNA contamination and re-concentrated
by RNeasy MinElute Cleanup Kit® (Qiagen), before being reverse-transcribed.
The enzyme, SuperScript® III (Invitrogen), was used in the mRNA - cDNA reverse
transcription in this and the following experiment. The mRNA expressions of
target genes were measured by Q-PCR with the Roche LightCycler® 480 System
(Roche Applied Science, Mannheim, Germany). PCRs contained 40 ng of cDNA,
50 nM of each primer, and the LightCycler® 480 SYBR Green I Master (Roche) in a
final volume of 10 μl. All Q-PCR reactions were performed as follows: 1 cycle of
50 ⁰C for 2 min and 95 ⁰C for 10 min, followed by 45 cycles of 95 ⁰C for 15 sec
and 60 ⁰C for 1 min using 0.05 μM of specific primers for stickleback GnRHs
20
(Table 3) PCR products were subjected to a melting-curve analysis, and
representative samples were electrophoresed to verify that only a single product
was present. Control reactions were conducted with RNA free water to
determine the background levels. The standard curve for each gene was
confirmed to be in a linear range with the reference gene (RPL8) while series
dilutions of cDNA as the template.
GtHs
Total RNA was extracted from stickleback’s pituitaries through
homogenization using TriZol reagent according to the methods in a previous
study (Hellqvist et al. 2008). The extracted RNA was treated with DNase (2U)
using TURBO DNA-free kit according to the manufacturer’s instruction (Ambion)
to remove possible genomic DNA contamination. SuperScript® III (Invitrogen),
was used in the mRNA - cDNA reverse transcription. After reverse-transcription,
the samples were diluted 1/10 for lh-β and fsh-β, or 1/100 for 18S
measurements. The relative quantity of lh-β, fsh-β and 18S mRNA expression
was measured by Q-PCR with the Stratagene MX3000P™ using Stratagene’s
MxPro™ Q-PCR Software with brilliant II Fast SYBR® Green Q-PCR Master Max
according to the manufacturer’s instructions (Stratgene, Agilent Technologies).
Specific primers for lh-β, fsh-βand 18s rRNA (0.05 μM) (Table 3) were used. PCRs
contained 40 ng (FSH-β, LH-β) or 4 ng (18s) of cDNA , 50 nM of each primer, and
same volume of Fast SYBR Green in a final volume of 10 μl.The parameters for
the real-time PCR were set at 95 ⁰C for 2 min followed by 40 cycles (at 95 ⁰C for 5
sec and 60 ⁰C for 20 sec). One last cycle was run to produce a melting curve to
show the amplified products specificity. lh-β and fsh-β mRNA expression data
were normalized against 18s as a reference gene.
21
Table 3. Sequences of the primers used in qRT-PCR. The RPL8 and 18S were used as reference genes in the measurement of cGnRH II / sGnRH and lh-β / fsh-βrespectively.
Gene Forward primer Reverse primer
cgnrh II 5’-CCGTCGGAGATTTCAGAGGAGATT-3’ 5’-TCTGAAGCTCTCTGGCTAAGGCAT-3’ (ENSGACG00000011943) sgnrh 5’-AGCGATGGTGCAGGTGTTGATGTT-3’ 5’-TTAAGTCTCTCTTGGGTCTGGGCA-3’ (ENSGACG00000012674) rpl8 5’-CGACCCGTACCGCTTCAAGAA-3’ 5’-GGACATTGCCAATGTTCAGCTGA-3’
(ENSGACG00000002035)
fsh-β 5’-CATCGAGGTGGAGGTCTGTG-3’ 5’-GGGGCTGATGGCTGCTGT-3’ (EMBL Accession No.AJ534871) lh-β 5’-GGTCACTGCCTCACCAAGGA-3’ 5’-GGAGCGCGATCGTCTTGTA-3’ (EMBL Accession No.AJ534969) 18S 5'-CTCAACACGGGAAACCTCAC-3' 5'-AGACAAATCGCTCCACCAAC-3'
(NCBI Accession No.BT026885.1)
In situ hybridization
Fragments of GnRH homologues were obtained by PCR and subcloned into the
pGEM-T easy vector. After the PCR with T7 and SP6 primers, digoxigenin
(DIG)-labeled (Perkin-Elmer, Boston, MA, USA) RNA antisense probes were
synthesized by in vitro transcription with proper (T7 or SP6) RNA polymerase (Roche,
Penzberg, Germany). RNA probes were examined by checking on commercial RNA
gels (Amresco, Solon, Ohio, USA).
Two sets of breeding males were used in Oct 2011 (n=2) and Jul 2012 (n=4). The
stickleback brains with pituitary were fixed with 4% paraformaldehyde (PFA) at 4 °C
for 3 h for cryosections (2011) or overnight for paraffin sectioning (2012). For
cryosection, the diencephalon (including pituitary) were separated from the brain
and immersed in phosphate buffer solution (PBS) containing 30% sucrose overnight,
and embedded with compound embedding medium (OCT) (Sakura, Tokyo, Japan) at
-20 °C. Transverse cryosections (about 10 μm) were made crossing the pituitary and
hypothalamus with a cryostat (CM 1900, Leica, Heidelberg, Germany), and were
attached to amino silane (APS) adhesive slides (Matsunami, Osaka, Japan). The
paraffin sections were made from the whole brain which was dehydrated following
the standard protocol, where DEPC treated water was used to wash the samples
before dehydration and to dilute alcohol in dehydration. Serial sagittal sections (5μm)
were made between c. 1.5mm right and left from the center line, which covered the
most of hypothalamus and telencephalon. The paraffin sections were attached to
APS adhesive slides. Prepared slides were washed with phosphate buffer solution
tween-20 (PBST) and then hybridized with probe at 65 °C for overnight incubation.
The concentration of DIG-labeled RNA probe examined by dot blot assay (control 100
22
ng/µL RNA as a reference) was about 750 ng/ml. On the next day, slides were
washed in a hybridization buffer series in 2x SSC at 65 °C. After another serial
washing with 0.2x SSC and PBST, samples were incubated with 5% sheep serum in 2
mg/mL bovine serum albumin (BSA) blocking solution. After the blocking step, slides
were incubated with an alkaline phosphatase (AP)-conjugated anti-DIG antibody
(Roche, 1:5,000 dilution in blocking solution) at 4 °C and then incubated in staining
buffer (0.1 M Tris , pH 9.5, 50 mM MgCl2, 0.1 M NaCl, and 0.1% Tween-20). Staining
was conducted with a mixture of nitrotetrazolium blue chloride (100 mg/mL) and
5-bromo-4-chloro-3-indolyl phosphate (BCIP) (50 mg/mL) in buffer. The reaction was
stopped with PBST and methanol. Images were obtained using a microscope
(Axioplan 2 Imaging, Carl Zeiss, Oberkochen, Germany).
Radioimmunoassay (RIA)
Each plasma sample was diluted with 100 μl RIA buffer (Schulz 1985) and
vortexed for 20 sec. Following heat treatment at 80 ⁰C for 60 min, the tubes were
centrifuged at 13000 rpm for 20 min in 4 ⁰C. The supernatants were pipetted into a
new set of tubes, which were stored in -70 ⁰C until measurement.
Both T and 11KT levels of the samples were measured via the
radioimmunoassay (RIA) developed by (Schulz 1985) to quantify levels directly from
heat-treated samples following the protocol in Schulz et al. (1994).
Statistics
The data were analysed with two tailed t-test for independent samples after
normality test (p<0.05) for comparisons between two groups and with one way
ANOVA for comparisons between multiple groups. The interaction effects between
treatment dosages and operations were analysed using two-way ANOVA by SPSS
v.14.
RESULTS AND DISCUSSION
GnRH gene expression in stickleback (paper I, II)
23
Both GnRH2 (cGnRH II) and GnRH3 (sGnRH) cDNA were identified in sticklebacks,
whereas there was no sign of any possible GnRH1 cDNA sequence. Via in situ
hybridization, the cells expressing GnRH3 were found in the hypothalamus and
telecephalon, but barely in other brain areas (Fig 7). The GnRH2 expressing neurons
were mainly located in the vental mesocephalon and thalamus, but they were found
in the hypothalamus as well (Fig 7). The distribution pattern of GnRHs neurons in the
stickleback brain was largely consistent with that of masu salmon (Oncorhynchus
masou) (review in Amano et al. 1997), whereas the GnRH2 expressing neurons were
not present in the hypothalamus of masu salmon. In Atlantic croaker (Micropogonias
undulatus), the hypothalamic GnRH-expressing neurons (GnRH1, sea bream GnRH)
and its mRNA level have also been detected in pituitary (Mohamed et al. 2005).
GnRH2 and 3 expressing cells were found in the pituitary of stickleback as well.
Figure 7. GnRHs expression in the brain of stickleback labeled via mRNA in situ hybridization. A. GnRH3 expressing cells in the whole brain sagittal section. B. GnRH3 expressing cells in the hypothalamus and pituitary, transverse section. C. GnRH2 expressing cells in the whole brain, sagittal section. D. GnRH2 expressing cells in the hypothalamus and pituitary, transverse section. Arrows indicated the expressing cells. T, telecephalon; OT, optic tectum-thalamus; C, cerebellum; Th, thalamus; HT, hypothalamus; D (-r/ -l), diencephalon (right/ left); POA, preoptic area; PIT, pituitary; MT, midbrain tegmentum; VT, ventral telencephalon. Bars = 600μm (A), (C); 300 μm (B), (D).
Dramatically higher expression levels of both gnrh2 and gnrh3 in breeding, than
24
in post-breeding sticklebacks, suggest that both gnrh2 and gnrh3 play roles in
stickleback reproduction. However, photoperiodic effects on GnRHs mRNA
expressions were less clear. There was a significantly higher expression level of
gnrh2 found under long day, but no difference was noticed in gnrh3 which appears
to be the dominant form in three-spined stickleback. Furthermore, in another study
the mRNA levels of gnrh3 in the stickleback has been found to be significantly
decreased, rather than increased, when the fish had been transferred to long day
photoperiod for 5 day (O’Brien et al 2012). The effects of light signal may influence
the BPG axis via other mechanism(s). Furthermore, the expression levels of gnrh2
and gnrh3 were regulated by gonadal feedbacks. Castration had inhibitory effect on
both gnrh2 and gnrh3 gene expressions in breeding males, whereas castration
casued stimulatory effect on gnrh3 expression in post-breeding males (Fig 8, this
result was not included in paper I).The polarity of the feedback effect on gnrh3 gene
expression may turn from positive to be negative when the males left the breeding
state.
Figure 8. GnRHs mRNA
levels in sham-operated
(n=9) or castrated (n=10)
post-breeding male
sticklebacks. Means±S.E.
shown, GnRH3: p < 0.05;
GnRH2: p=0.324.
The mRNA levels of lh-β, fsh-β or gnrh2, gnrh3 were not suppressed in
stickleback infected with S. solidus, but rather the contrary. The results indicated
that suppression of gonads and secondary sexual characters in stickleback was not
due to an inhibition by parasite on the brain-pituitary level. These results are in
contrast to the effects found in the common roach (Rutilus rutilus), where the lh-β
mRNA levels, and also pituitary LH content, were diminished in Ligula intestinalis
infected fish (Carter et al. 2005). Trubiroha et al. (2009) found a suppressive effect of
Ligula on both lh-β and fsh-β mRNA in both sexes. The mechanism which caused the
25
increase of the expressions of GtHs and GnRHs in infected stickleback is still unclear.
Since the infected males may have lower androgen levels than uninfected males
(MacNab et al. 2011), this stimulatory effects may be due to the feedback on the
host’s BPG axis.
However, how the parasite infection up-regulated GnRHs and GtHs gene
expressions is still unclear. This study suggested that the stimulatory effects on
sexual endocrine system in the brain and pituitary levels may be one of the
mechanisms to compensate the impacts of parasite, which provide sticklebacks the
potential to mature, at least at a minimum level.
Feedback control of GtH gene expression (paper III)
The sham-operated fish kept under long day (LD 16:8) have higher levels of lh-β
mRNA and fsh-β mRNA than those kept under under short day (LD 8:16). The
negative feedback effects were more marked under short days than under long days,
whereas positive feedback could be more obvious under long days (Table 9). Under
LD 8:16, low doses of T suppressed both lh-β and fsh-β mRNA. However, with high
doses of T treatments or under longer photoperiods, the negative effects on lh-β and
fsh-β mRNA often diminished or turned to be positive (Table 9). In mammals and
birds, the photoperiod can influence the strength of feedback effect on GtH
(Kriegsfeld and Bittman 2010, Wilson 1984), whereas it does not alter the polarity of
feedback. In those cases, the inhibitory photoperiod can enhance the negative
feedback in both ewe (Rosa & Bryant 1985) and male hamsters (Turek 1977).
The inhibitory effect of low T on lh-β and fsh-β under LD 8:16 were abolished by
AI, whereas the stimulatory effect of high T was not, suggesting that estrogen may
play a role in suppressing male maturation under short day. This is consistent with
that the feedback effects of the non-aromatisable androgen 11KA are similar to T
cryst both in the present and in previous studies (Hellqvist et al. 2008). However,
those results were in contrast to the extensively studied positive feedback effects in
salmonids, where previous studies have shown that pituitary LH levels in salmonids
can be stimulated by aromatizable androgens and estrogens (Crim et al. 1981). A
26
positive feedback has also been found in European eel pituitary cells in vitro, where
the GtH II (LH) content and GtH II – β (lh-β) mRNA level increased with treatments
both with T and the non-aromatizable androgen dihydrotestosterone (DHT), but not
with estrogen (Huang et al. 1997).
Table 9. Effect of photoperiod, operation and implants on GtH mRNA levels feedbacks
Year Comparison Photoperiod (L:D) lh-β feedback# fsh-β feedback#
2007 Sham vs. Cast. 11:13 0.15 * 0.04 **
12:12 9.32 *** 0.07 **
13:11 53.57 *** 0.32 *
14:10 2.54 ** 0.36 *
Cast. vs. T0.25 11:13 0.27 * 0.15 *
12:12 0.29 * 0.26 *
13:11 0.22 * 0.62 ns
14:10 0.21 * 2.06 ns
2008 Sham vs. Cast. 8:16 / 16:8 0.26 * / 19.51 * 0.05 * / 0.71ns
Cast. vs. T0.25 8:16 / 16:8 0.06 ** / 8.4 ns 0.02 ** / 0.27 *
Cast. vs. Tcryst 8:16 / 16:8 5.12 * / 26.33 ** 0.02 ** / 2.09 ns
Cast. vs. 11KA 8:16 / 16:8 1.06 ns / 20.15 * 0.11 * / 2.11ns
2010 Sham vs. Cast. 8:16 0.33 * 0.12 *
Cast. vs. T0.25 8:16 0.23 * 0.16 **
Cast. vs. Tcryst 8:16 2.08 * 0.18 *
Cast. vs. T0.25 8:16 0.83 ns 0.35 *
+ AI
Cast. vs. Tcryst 8:16 1.86 * 0.23 *
+ AI
Cast. vs. AI 8:16 0.32 * 0.68 *
2011 Sham vs. Cast. 8:16 / 16:8 0.50 * / 5.67 * 0.26 * / 2.88 **
Cast. vs. T0.10 8:16 / 16:8 0.30 * / 0.39 ns 0.08 * / 0.14 *
Cast. vs. T0.25 8:16 / 16:8 0.29 * / 0.34 ns 0.08 * / 0.12 *
Cast. vs. T0.50 8:16 / 16:8 0.18 ** / 2.11 * 0.18 * / 0.63 ns
Castration (Cast.), Sham-operated (Sham) Feedback#: The mRNA levels of sham operated fish are compared to the mRNA levels of castrated control fish in the same photoperiod and experiment. The castrated controls are set to 1 and thus ratio <1 indicates negative feedback and ratios >1 indicate positive feedback.. Implants: Control (empty or cacaobutter alone), T cryst (Crystalline testosterone), T 0.10/ 0.25/ 0.50 (0.10%/0.25%/0.50% T in cacaobutter), 11KA (Crystalline 11-ketoandrostenedione), AI (aromatase inhibitor). ns not significan; * p<0.05; ** p<0.01; *** p<0.001.
27
The suppression of both lh-β and fsh-β by low androgen levels, especially under
short day, may inhibit maturation completely unless a rise of androgens above a
threshold level would allow complete maturation (Fig 8).
Figure 9. The proposed model of the polarity shift in feedback effect. The androgen
threshold for positive feedback decreased as the light hours increasing. Then, the androgens
feedback may turn from negative to be positive under long photoperiod.
Homeostasis of circulating androgens levels (paper IV)
Androgens plasma levels in the hemi-castrated fish were only half of the levels
in sham-operated fish, which showed that there was no compensatory
steroidogenesis from the remaining testis, which is in agreement with Hellqvist et al.
(2002).
28
Lower doses of 11KA and T implants increased plasma androgen levels in hemi-
or completely castrated but not in sham-operated fish. Both low and high doses of
11KA increased plasma 11KT levels in hemi-castrated fish, whereas only the high
dose of 11KA did so in sham-operated fish. Kurtz et al. (2007) implanted intact
breeding stickleback males with high and low doses of 11KA. In contrast to our
results, plasma 11KT rose significantly after the treatment of both dosages. The
difference may be due to that they used larger capsules and smaller fish, thus their
low dose was probably in effect higher than the one we used.
The circulating GtH levels may increase after castration in breeding fish,
indicating a negative feedback (Schulz et al 2012). However, not only negative but
also positive feedbacks on the BPG axis are known from fishes, including the
stickleback (Hellqvist et al. 2008). Steroidogenesis in testis of male stickleback can be
suppressed by treating the fish with a high dose of methyltestosterone via the water
(Borg et al. 1985). In the present study, it is doubtful if androgen-treatments led to a
dramatic decrease in steroid production, since 11KT levels were not changed by T
treatments and T levels not by 11KA treatments. However, the homeostatic control
may be exerted not only by androgen-treated fish producing less androgens in the
testes but perhaps also by higher clearance of circulating androgens.
In case aromatization plays a role in homeostatic mechanisms, androgen levels
would be expected to rise in fish treated with inhibitors. However, this was not the
case. Reduced plasma androgen levels in hemi-castrated fish were not compensated
in fully mature male stickleback, which suggested that the remaining testis’
steroidogenic activity had been fully activated already. However, both 11KA and T
treatments increased plasma levels much less in sham-operated fish than in
castrated ones, indicating that homeostatic mechanisms dampen increases of both
11KT and T plasma level.
Summary
Both GnRH2 (cGnRH II) and GnRH3 (sGnRH) neurons were present in the
stickleback’s hypothalamus, and may be involved in the seasonal reproduction.
29
There was no evidence for a role of gnrh3 in the photoperiodic control of
reproduction. However, the polarity of the feedback effect on gnrh3 gene
expression may turn from positive to be negative when the males left the
breeding state.
S. solidus infection can up-regulate both GnRHs and GtHs gene expressions,
which may be due to feedback effects on the BPG axis.
The polarity of feedback on lh-β and fsh-β was regulated by both photoperiod
and plasma sex steroids levels.
The suppression of both lh-β and fsh-β by low androgen levels, especially under
short day, may inhibit maturation completely unless a rise of androgens above a
threshold levels would allow complete maturation. This may cause all or
nothing maturation.
The androgen inhibitory effect on lh-β and fsh-β under short day could be
abolished by aromatase inhibitor, which may explain how estrogen suppresses
male maturation under short day.
The reduction of plasma androgen levels in fully mature hemi-castrated fish may
suggest that the remaining testis is not able to increase its steroidogenesis
further. However, both 11KA and T treatments increased plasma levels much
less in sham-operated fish than in castrated ones, indicating that homeostatic
mechanisms are nevertheless present.
30
ACKNOWLEDGEMENTS
I would like to thank my supervisor Bertil Borg for his excellent scientific guidance,
and my co-supervisor, Dick Nässel, my committees, Mention Sören Nylin and Bodil
Elmhagen ,and Heinrich Dircksen for discussions and comments. Your advices are
always valuable. I also thank my coauthors, Monika Schmitz and Susanne Trombley
in Uppsala University, and Pung Pung Hwang and Yung Che Tseng in Academia Sinica
for kind hospitality and guidance, and Rüdiger Schulz and Wytschke van Dijk for the
helps in experiments and writing. Thanks Jaingnan Lou, Oleh Lushchak, Neval Kapan
and Erik Hoffmann, my friends, for always being there whenever I needed your help.
I would also like to thank people at the department, Anette Lorents, Minna
Miettinen, Ulf Norberg, Berit Strand and Siw Gustafsson for making the environment
pleasant to be and work in. The former people at the department, Johannes Strauss,
Agata Jolodziejczyk, Jeannette Söderberg, Lina Enell, Lily Tesfai, Kristin Eriksson,
thank you for your kindness and help. Always! I would like to thank all of the
“customers” of our Golden Stickleback pub. We enjoyed many wonderful Friday
afternoons sharing beers and dreams.
I am thankful to all of my friends in Taiwan and Sweden, for always encouraging and
helping me. I also want to thank my family, especially my wife, Evy, my study could
not have been finished without you.
31
REFERENCES
Aalbers SA (2008) Seasonal, diel, and lunar spawning periodicities and associated
sound production of white seabass (Atractoscion nobilis). Fish Bull 106: 143-151.
Almeida FFL, Taranger GL, Norberg B, Karlsen Ø , Bogerd J, Schulz RW (2009)
Photoperiod-Modulated Testis Maturation in Atlantic Cod (Gadus morhua, L.).
Biol Reprod 80: 631–640
Amano M, Hyodo S, Kitamura S, Ikuta K, Suzuki Y, Urano A, Aida K (1995) Short
photoperiod accelerates preoptic and ventral telencephalic salmon GnRH
synthesis and precocious maturation in underyearling male masu salmon. Gen
Comp Endocrinol 99:22–27.
Amano M, Urano A, Aida K (1997) Distribution and function of
gonadotropin-relesasing hormone (GnRH) in teleost brain. Zool Sci 14:1-11.
Andersson E, Bogerd J, Borg B, Sharp P J, Sherwood NM, Goos HJTh (1995)
Characterization and localization of gonadotropin-releasing hormone in the
brain and pituitary of the three-spined stickleback, Gasterosteus aculeatus.
Cell Tissue Res 279:485-494.
Andersson E, Borg B, Goos HJTh (1992) Temperature, but not photoperiod, influences
gonadotropin-releasing hormone binding in the pituitary of the three-spined
stickleback, Gasterosteus aculeatus. Gen Comp Endocrinol 88:111-116.
Antonopoulou E, Mayer I, Berglund I, Borg B (1995) Effects of aromatase inhibitors on
sexual maturation in Atlantic salmon, Salmo salar, male parr. Fish Physiol
Biochem 14:15-24
Baggerman B (1957) An experimental study on the timing of breeding and migration
in the three-spined stickleback (Gasterosteus aculeatus L.). Arch Neerl Zool
12:105-318.
Baggerman B (1985) The role of biological rhythms in the photoperiodic regulation of
seasonal breeding in the stickleback Gasterosteus aculeatus. Neth J Zool
35:14– 31.
32
Baggerman B (1989) On the relationship between gonadal development and
response time to photostimulation of sticklebacks living under natural
conditions and under constant short-day conditions for long periods of time. Can
J Zool 67:126– 135.
Barber I (2007) Host-parasite interactions of the Three-spined stickleback. In:
Huntingford, F.A., Ö stlund-Nilsson, S., Mayer, I. (Eds.) Biology of the
Three-spined Stickleback. CRC press, pp 271-318.
Begtashi I, Rodriguez L, Moles G, Zanuy S, Carrillo M (2004) Long-term exposure to
continuous light inhibits precocity in juvenile male European sea bass
(Dicentrarchus labrax, L.). I. Morphological aspects. Aquaculture 241: 539–559.
Billard R, Breton B (1978) Rhythms of reproduction in teleost fish. In: Thorpe, J. E.
(Ed.) Rhythmic Activity of Fishes. Academic Press, pp. 31-53.
Billard R, Richard M, Breton B (1977) Stimulation of gonadotropin secretion after
castration in rainbow trout. Gen Comp Endocrinol 33: 163-165.
Bogerd J, Zandbergen T, Andersson E, Goos H (1994) Isolation, characterization and
expression of cDNAs encoding the catfish-type and chicken-II-type
gonadotropin-releasing hormone precursors in the African catfish. Eur J
Biochemi 222: 541–549.
Borg B (1982) Seasonal effects of photoperiod and temperature on spermatogenesis
and male secondary sexual characters in the threespined stickleback,
Gasterosteus aculeatus. Can J Zool 60:3377–3386.
Borg B (2007) Reproductive physiology of sticklebacks, In: S. Ö stlund-Nilsson, Mayer,
I., Huntingford, F.A. (Eds.) Biology of the Three-spined Stickleback. CRC press,
pp. 225-248.
Borg B (2010) Photoperiodism in fishes. In: R. J. Nelson, Denlinger D.L., Somers D.E.
(Eds) Photoperiodism: The Biological Calender. Oxford University Press. pp
371-398.
Bromage NR, Jones J, Randall CF, Thrush M, Davies B, Springate J, Duston J, Barker G
(1992) Broodstock management, fecundity, egg quality and timing of egg
33
production in the rainbow trout (Oncorhynchus mykiss). Aquaculture 100:
141–166.
Bromage N R, Randall CF, Duston J, Thrush M, Jones J (1994) Environmental control of
reproduction in salmonids. In: Muir, J. F. and Roberts, R. J. (Eds.) Recent advances
in Aquaculture IV. Blackwell, pp. 55-65.
Burgus R, Butcher M, Amoss M, Ling N, Monahan M, Rivier J, Fellows R, Blackwell R,
Vale W, Guillemin R (1972) Primary structure of ovine luteinizing
hormone-releasing factor (LRF). Proc Nat Acad Sci USA 69:278–282.
Borg B, Reschke M, Peute J, van den Hurk R (1985) Effects of castration and
androgen-treatment on pituitary and testes of the three-spined stickleback,
Gasterosteus aculeatus L., in the breeding season. Acta. Zool. (Stockh.) 66:47-54.
Fernald RD, White RB (1999) Gonadotropin-releasing hormone genes: Phylogeny,
structure, and functions. Front Neuroendocrin 20:224–240.
Carter V, Pierce R, Dufour S, Arme C, Hoole D (2005) The tapeworm Ligula intestinalis
(Cestoda: Pseudophyllidea) inhibits LH expression and puberty in its teleost
host, Rutilus rutilus. Reproduction 130:939-945.
Crim LW, Evans DM (1979) Stimulation of pituitary gonadotropin by testosterone in
juvenile rainbow trout (Salmo gairdneri). Gen Comp Endocrinol 37: 192-196.
Crim LW, Peter RE, Billard R (1981) Onset of gonadotropic hormone accumulation in
the immature trout pituitary gland in response to estrogen or aromatizable
androgen steroid hormones. Gen Comp Endocrinol 44:374-381.
Davie A, Porter MJR, Bromage NR (2003) Photoperiod manipulation of maturation
and growth of Atlantic cod (Gadus morhua). Fish Physiol Biochem 28:399-401.
Dubinina MN (1980) Tapeworms ( Cestoda, Ligulidae ) of the fauna of the USSR.
(Online version, translated from the Russian text of 1966). New Delhi: Amerind
Publishing Co., 320 pp.
Endal HP, Taranger GL, Stefansson SO, Hansen T (2000) Effects of continuous
additional light on growth and sexual maturity in Atlantic salmon, Salmo salar,
34
reared in sea cages. Aquaculture 191: 337–349.
Goos HJTh (1991) Fish gonadotropins and GnRH’s: fundamental aspects and practical
applications. Bulletin of the Institute of Zoology, Academia Sinica, Monograph.
16: 119-137.
Goos HJTh, Joy KP, De Leeuw R, Van Oordt PGWJ, Van Delft AML, Gielen JTh. (1987)
The effect of luteinizing hormone-releasing hormone analogue (LHRHa) in
combination with different drugs with anti-dopamine and anti-serotonin
properties on gonadotropin release and ovulation in the African catfish,
Clarias gariepinus. Aquaculture 63: 143-156.
Hansen T, Karlsen Ø , Taranger GL, Hemre GI, Holm JC, Kjesbu OS (2001) Growth,
gonadal development and spawning time of Atlantic cod (Gadus morhua)
reared under different photoperiods. Aquaculture 203:51-67.
Huang YS, Schmitz M, Belle NL, Chang CF, Quérat B, Dufour S (1997) Androgens
stimulate gonadotropin-II β-subunit in eel pituitary cells in vitro. Mol Cell
Endocrinol 131: 157-166.
Heins DC, Baker JA (2003) Reduction of egg size in natural populations of threespined
stickleback infected with a cestode macroparasite. J Parasitol. 89: 1-6.
Heins DC, Baker JA, Toups MA, Birden EL (2010) Evolutionary significance of
fecundity reduction in threespine stickleback infected by the
diphyllobothriidean cestode Schistocephalus solidus. Biol J Linn Soc
100:835-846.
Hellqvist A, I Mayer, B Borg (2002) Effect of hemi-castration on plasma steroid levels
in two teleost fishes; the three-spined stickleback, Gasterosteus aculeatus, and
the Atlantic salmon, Salmo salar. Fish Physiol Biochem 26:107-110.
Hellqvist A, Schmitz M, Borg B (2008) Effects of castration and androgen-treatment
on the expression of FSH-β and LH-β in the threespine stickleback,
Gasterosteus aculeatus –feedback differences mediating the photoperiodic
maturation response? Gen Comp Endocrinol 158:678-682.
Hellqvist A, Schmitz M, Mayer, Borg B (2006) Seasonal changes in expression of LH-β
35
and FSH-β in male and female three-spined stickleback, Gasterosteus
aculeatus. Gen Comp Endocrinol 145:263-269.
Hoover EE, Hubbard HE (1937) Modification of the sexual cycle in trout by control of
light. Copeia 4:206-210.
Howell RA, Berlinsky DL, Bradley TM (2003) The effects of photoperiod manipulation
on the reproduction of black sea bass, Centropristis striata. Aquaculture
218:651–669.
Jakobsson S, Borg B, Haux C, Hyllner SJ (1999) An 11-ketotestosterone induced
kidney-secreted protein: the nest building glue from male three-spined
stickeback, Gasterosteus aculeatus. Fish Physiol Biochem 20:79-85.
King JA, Dufour S, Fontaine YA, Millar RP (1990) Chromatographic and immunological
evidence for mammalian GnRH and chicken GnRH II in eel (Anguilla anguilla)
brain and pituitary. Peptides 11:507–514.
King JA, Millar RP (1982) Structure of chicken hypothalamic luteinizing
hormone-releasing hormone II. Isolation and characterization. J Biol Chem
257:10729–10732.
Kriegsfeld LJ, Bittman EL (2010) Photoperiodism in mammals. In: R.J. Nelson,
Denlinger D.L., Somers D.E. (Eds), Photoperiodism: The Biological Calender,
Oxford University Press., pp 462-503.
Kurtz J, Kalbe M, Langefors Å, Mayer I, Milinski M, Hasselquist D (2007) An
experimental test of the immunocompetence handicap hypothesis in a teleost
fish: 11-ketotestosterone suppresses innate immunity in three-spined
sticklebacks. Am Nat 170:509-519.
Lin XM, Peter RE (1996) Expession of salmon gonadotropin-releasing hormone
(GnRH) and chicken GnRH-II precursor messenger ribonucleic acid in the brain
and ovary of goldfish. Gen Comp Endocrinol 101:282–296.
MacNab V, Katsiadaki I, Barber I (2009) Reproductive potential of Schistocephalus
solidus infected male three-spined stickleback Gasterosteus aculeatus from
two U.K. populations. J Fish Biol 75:2095-2107.
36
MacNab V, Scott AP, Katsiadaki I, Barber I (2011) Variation in the reproductive
potential of Schistocephalus infected male sticklebacks is associated with
11-ketotestosterone titre. Horm Behav 60:371-379.
Maugars G, Schmitz M (2008) Gonadotropin and gonadotropin receptor expression
during the onset of sexual maturation in early maturing Atlantic salmon male
parr, Salmo salar. Mol Reprod Dev 75:403-413.
Mayer I, Borg B, Schulz R (1990) Seasonal changes in and effect of
castration/androgen replacement on the plasma levels of five androgens in
the male three-spined stickleback, Gasterosteus aculeatus L. Gen Comp
Endocrinol 79:23-30.
McPhail JD, Peacock SD (1983) Some effects of the cestode (Schistocephalus solidus)
on reproduction in the threespine stickleback (Gasterosteus aculeatus):
evolutionary aspects of a host-parasite interaction. Can J Zool 61:901.
Miranda LA, Montaner AD, Ansaldo M, Affanni JM, Somoza GM (1999)
Characterization of brain gonadotropin-releasing hormone (GnRH) molecular
variants in brain extracts from different perciform fishes from Antarctic waters.
Polar Biol 21:122-127.
Miyamoto K, HasegawaY, Nomura M, Igarashi M, Kangawa K, Matsuo H (1984)
Identification of the second gonadotropin releasing hormone in chicken
hypothalamus: Evidence that gonadotropin secretion is probably controlled by
two distinct gonadotropin-releasing hormones in avian species. Proc Nat Acad
Sci USA 81: 3874–3878.
Mohamed JS, Thomas P, Khan IA (2005) Isolation, cloning, and expression of three
prepro-GnRH mRNAs in Atlantic croaker brain and pituitary. J Comp Neurol
488:384–395.
Montaner AD, Miranda LA, Vizziano D, López A, Okuzawa K, Somoza GM (2001)
Gonadotropin-releasing hormone in two perciform fishes: Micropogonias
furnieri and Pagrus pagrus. Fish Physiol Biochem 24:243-246.
37
Morehead DT, Ritar AJ, Pankhurst NW (2000) Effect of consecutive 9- or 12-month
photothermal cycles and handling on sex steroid levels, oocyte development
and reproductive performance, in female striped trumpeter Latris lineate
(Latrididae). Aquaculture 189: 293–305.
Ngamvongchon S, Lovejoy DA, Fischer WH, Craig AG, Nahorniak CS, Peter RE, Rivier
JE, Sherwood NM (1992) Primary structures of two forms of
gonadotropin-releasing hormone, one distinct and one conserved, from
catfish brain. Mol Cell Neurosci 3: 7–22.
O'Brien CS, Bourdo R, Bradshaw WE, Holzapfel CM, Cresko WA (2012) Conservation
of the photoperiodic neuroendocrine axis among vertebrates: Evidence from
the teleost fish, Gasterosteus aculeatus. Gen Comp Endocrinol 178: 19-27
Okubo K, Nagahama Y (2008) Structural and functional evolution of
gonadotropin-releasing hormone in vertebrates. Acta Physiologica 193: 3–15.
Palevitch O, Kight K, Abraham E, Wray S, Zohar Y, Gothilf Y (2007) Ontogeny of the
GnRH systems in zebrafish brain: in situ hybridization and promoter-reporter
expression analyses in intact animals. Cell Tissue Res 327:313–322.
Pauly D (1998) Tropical fishes: patterns and propensities. J Fish Biol 53:1-17.
Perdikaris C (2001) Photoperiodic control of early maturation. Applicability of the
method to Mediterranean seabass (Dicentrarchus labrax) and seabream (Sparus
aurata) culture. 10th Proc. Panell. Congr. Ichthyol. pp: 161-164.
Peter RE, Trudeau VL, Sloley BD (1991) Brain regulation of reproduction in teleosts.
Bulletin of the Institute of Zoology, Academia Sinica, Monograph 16: 89-118
Petersen CW, Warner RR (2002) The ecological context of reproductive behavior. In:
Sale P. F. (Ed) Coral Reef Fishes. Academic Press, pp. 103-120.
Porter MJR, Duncan NJ, Mitchell D, Bromage NR (1999) The use of cage lighting to
reduce plasma melatonin in Atlantic salmon Salmo salar and its effects on the
inhibition of grilsing. Aquaculture 176:237–244.
Randall CF, Bromage NR, Duston J, Symes J (1998) Photoperiod induced phase-shifts
38
of the endogenous clock controlling reproduction in the rainbow trout: a
circannual phase-response curve. J Reprod Fertil 112:399-405.
Rosa HJD, Bryant MJ (2003) Seasonality of reproduction in sheep. Small Ruminant
Res 48:155-171.
Schulz R (1985) Measurement of five androgens in the blood of immature and
maturing male rainbow trout, Salmo Gairdneri (Richardson). Steroids 46:717-726.
Schultz ET, Topper M, Heins DC (2006) Decreased reproductive investment of female
threespined stickleback Gasterosteus aculeatus infected with cestode
Schistocephalus solidus: parasite adaptation, host adaptation, or side effect?
Oikos 114: 303-310.
Schulz RW, W van Dijk, E Chaves-Pozo, Á García-López, LR de França, J Bogerd (2012)
Sertoli cell proliferation in the adult testis is induced by unilateral
gonadectomy in African catfish. Gen Comp Endocrinol 177:160-167.
Sherwood NM, Eiden L, Brownstein M, Spiess J, Rivier J, Vale W (1983)
Characterization of a teleost gonadotropin-releasing hormone. Proc Nat Acad
Sci USA 80:2794–2798.
Somoza GM, Miranda LA, Strobl-Mazzulla P, Guilgur LG (2002) Gonadotropin-
releasing hormone (GnRH): from fish to mammalian brains. Cell Mol Neurobiol
22:589-609.
Stacey NE (1984) Control of the timing of ovulation by exogenous and endogenous
factors. In: Potts, G.W., Wootton, R.J. (eds) Fish reproduction: strategies and
tactics. Academic Press pp. 207-222.
Steven C, Lehnen N, Kight K, Ijiri S, Klenke U, Harris WA, Zohar Y (2003) Molecular
characterization of the GnRH system in zebrafish (Danio rerio): cloning of
chicken GnRH-II, adult brain expression patterns and pituitary content of
salmon GnRH and chicken GnRH-II. Gen Comp Endocrinol 133:27–37.
Suzuki M, Hyodo S, Kobayashi M, Aida K, Urano A (1992) Characterization and
localization of mRNA encoding the salmon-type gonadotropin-releasing
hormone precursor of the masu salmon. J Mol Endocrinol 9:73–82.
39
Tsutsui K, Ubuka T, Bentley GE, Kriegsfeld LJ (2012) Gonadotropin-inhibitory hormone
(GnIH): Discovery, progress and prospect. Gen Comp Endocrinol 177:305-314.
Taranger GL, Haux C, Stefansson SO, Björnsson BT, Walther B, Hansen T (1998) Abrupt
changes in photoperiod affect age at maturity, timing of ovulation and plasma
testosterone and oestradiol-17β profiles in Atlantic salmon, Salmo salar.
Aquaculture 162: 85-98.
Tostivint H (2011) Evolution of the gonadotropin-releasing hormone (GnRH) gene
family in relation to vertebrate tetraploidizations. Gen Comp Endocrinol
17:575-581.
Trubiroha A, Wuertz S, Frank SN, Sures B, Kloas W (2009) Expression of gonadotropin
subunits in roach (Rutilus rutilus, Cyprinidae) infected with plerocercoids of
the tapeworm Ligula intestinalis (Cestoda). Int J Parasotol 39:1465-1473.
Turek FW (1977) The interaction of photoperiod and testosterone in regulating
serum gonadotropin levels in castrated male hamsters. Endocrinology
101:1210-1215.
Yu KL, Sherwood NM, Peter RE (1988) Differential distribution of two molecular
forms of gonadotropin-releasing hormone in discrete brain areas of goldfish
(Carassius auratus). Peptides 9: 625–630.
Voet D, Voet J (2004) Biochemistry, 3rd ed. Wiley. USA.
Wilson FE (1985) Androgen feedback-dependent and –independent control of
photoinduced LH secretion in male tree sparrows (Spizella arbora). J Endocrinol
105:141-152.
Withler RE, Clarke WC, Blackburn J, Baker I (1998) Effect of triploidy on growth and
survival of pre-smolt and post-smolt coho salmon (Oncorhynchus kisutch).
Aquaculture 168: 413-422.
Wu SC, Horng JL, Liu ST, Hwang PP, Wen ZH, Lin CS, Lin LY (2009)
Ammonium-dependent sodium uptake in mitochondrion-rich cells of medaka
(Oryzias latipes) larvae. Am J Physiol-Cell Ph 298:237-250