Characterization of the Brain-pituitary axis in pejerrey Odontesthes bonariensis
G.M. Somoza, L.A. Miranda, L.G. Guilgur, and P.H. Strobl-Mazzulla
Laboratorio de Ictiofisiología y Acuicultura. IIB-INTECH. CC 164. (B7130IWA). Chascomús, provincia de Buenos Aires, Argentina.
Address correspondence to: Gustavo M. Somoza. IIB-INTECH, Camino de Circunvalación Laguna, Km 6 (B7130IWA) Chascomús, ARGENTINA. E-mail: somoza@intech.gov.ar
Key words: Brain; Pituitary; Reproduction; Sex differentiation.
The pejerrey fish (Odontesthes bonariensis), a large
atherinid native from the inland waters of Buenos Aires
Province, is considered one of the most emblematic fresh
water fish species of Argentina (López et al., 2001).
Although it is an important commercial and game fish,
its culture has not been well developed in Argentina.
Besides, some reports have demonstrated the existence
of problems in intensive pejerrey aquaculture: early
sexual maturation, the presence of spawning asynchrony
between females and low growth rates (Strüssmann,
1989; Strüssmann et al., 1993).
During the last years the interest of the laboratory
has been focussed on the study of the reproductive physiology
and the mechanisms involved during sexual determination/
differentiation in pejerrey, not only from a
basic point of view but also keeping attention to the
potential application of the knowledge on the development
of pejerrey aquaculture.
In this context, the goal of the present work was to
summarize the findings on the characterization of the
brain-pituitary-gonadal axis in Odontesthes bonariensis.
Gonadotropin-releasing hormone
Gonadotropin-releasing hormone (GnRH) is a key
neurohormone that regulates reproduction in all groups
of vertebrates. This decapeptide is synthesized by neurons
in different regions of the brain being its best known
function the regulation of gonadotropins production and
release by the pituitary gland (Seeburg et al., 1987).
Today it is well known that the brain of vertebrates
expresses at least two GnRH variants. However, in teleosts
fish there is a growing number of species in which
three different GnRH forms have been identified
(Somoza et al., 2002a, b; Lethimonier et al., 2004). Up
to date 14 different GnRH forms have been described
in vertebrates and they are usually called using the common
name of the species in which they were first described
(Adams et al., 2002; Lethimonier et al., 2004).
In those bony fish species expressing three variants in
their brain, these GnRH forms are distributed over specific
brain areas: chicken GnRH-II (cGnRH-II) is expressed
by neurons of the midbrain tegmentun (MT),
salmon GnRH (sGnRH) is localized in the terminal
nerve ganglion (TNG) and the third species-specific
form is mainly found in the anterior preoptic area (POA)
and the hypothalamus (Fernald and White 1999;
González-Martínez et al., 2001).
Also in pejerrey fish, three different GnRH variants
have been found: sGnRH, cGnRH-II, and pejerrey
GnRH (pjGnRH), a novel member of the GnRH family
isolated for the first time in this species (Stefano et al.,
1997; Montaner et al., 2001). The immunostaining of
pejerrey brain sections showed that sGnRH is mainly
expressed by neurons located at the TNG, pjGnRH at
the nucleus preopticus periventricularis (NPP) of the
POA, and cGnRH-II at the MT (Stefano et al., 2000).
In addition, only pjGnRH was detected in fibers entering
the anterior pituitary gland (Stefano et al., 1997,
2000; Somoza et al., 2002a). More recently the full
length of the cDNAs encoding for the three GnRH precursors
of pejerrey was obtained by the RACE method
(Rapid Amplification of cDNA Ends). The analysis of
these sequence showed that the cDNAs of pjGnRH,
cGnRH-II and sGnRH have 441, 530 and 516 bp, having
an open reading frame of 297, 252 and 276 bp, respectively
(Guilgur et al., 2003; GenBank accession
#AY744689, #AY744687, #AY744688). It was also
demonstrated the expression of different GnRH forms
in other pejerrey organs such as: gonads, eye, kidney,
spleen, liver, gill and olfactory epithelium suggesting
novel functions for these peptides (Guilgur et al., 2003).
When GnRH precursors from pejerrey are subjected
to a phylogenetic analysis they can be grouped following
the scheme proposed by Vickers et al. (2004):
pjGnRH in group one (GnRH I), containing GnRH variants
located at the POA and related to the control of the
pituitary gland; cGnRH-II from pejerrey in group two
(GnRH II), located in neurons of the MT and sGnRH
from pejerrey in group three (GnRH III), in neurons of
the anterior brain (Fig. 1). In this framework it is also
important to note the high percentages of identity observed
between pjGnRH and seabrean GnRH (sbGnRH)
precursors of different Acanthopterigian species, reinforcing
the hypothesis that pjGnRH emerged from the
gene codifying for sbGnRH (Montaner et al., 2001;
Somoza et al., 2002b).
FIGURE 1. Phylogenetic tree of GnRH preprohormones from different teleost species. The tree was constructed using a Clustal V multiple sequence alignment program (DNAstar). The deduced amino acid sequences were obtained from the GenBank.
Gonadotropins
Gonadotropins (GtHs) are pituitary heterodimeric
glycoproteins consisting of a common a-subunit and a b-subunit that confers hormonal specificity. Teleost fish
GtHs are structurally related to the tetrapod folliclestimulating
(FSH) and luteinizing (LH) hormones and
are also important in the regulation of gametogenesis
and sexual maturation (Quérat et al., 2000; Swanson et
al., 2003).
In the last years, the genes codifying for both gonadotropins
have been cloned and their expression studied
in many bony fish species (see Weltzein et al., 2004).
As a first step to study the function of GtHs in pejerrey,
the full length of the cDNAs encoding for FSH and LH
-b subunits were obtained by RACE (Miranda et al.,
2003a; 2004). The cDNA for FSH-SS and LH-SS have
466 and 558 bp with an open reading frame of 351 and
450 bp, respectively (Miranda et al., 2004). Comparing
these sequences with the deduced aminoacidic sequence
of other teleost species it can be inferred that pejerrey
FSH-b has a signal pepide of 15 amino acids (aa), and a
mature peptide of 102 aa with a putative N-linked
glycosylation site at residue Asn 12. Pejerrey LH-b is
composed by a signal peptide of 32 aa, a mature peptide
of 118 aa and a putative N-linked glycosylation site
at residue Asn 10. Pejerrey LH-b, as well as all vertebrate
species, has conserved the position of 12 cysteines
and 1 putative N-linked glycosylation site (Swanson et
al., 2003; Weltzein et al., 2004). Pejerrey FSH-b, also
contains 12 cysteines; however, the position and number
of cysteines in FSH-b varied among teleosts
(Swanson et al., 2003).
The aminoacid sequence of pejerrey FSH-b compared
to other teleost fish sequences varies between 33.1- 66.9% and is lower than that observed for LH-b subunits
(46.3-72.4%). These results follow the pattern indicating
that the primary structure of LH-b subunits has been better
conserved than FSH-b during teleosts evolution, suggesting
a rapid divergence of the FSH-b subunit (Quérat et al., 2000). Comparison studies also showed that
pejerrey GtHs are closer to other Actinopterigian GtHs
(Fig. 2).
FIGURE 2. Phylogenetic tree of b-GtHs subunits from different teleost species. The tree was constructed using a Clustal V multiple sequence alignment program (DNAstar). The deduced amino acid sequences were obtained from the GenBank.
Sex differentiation
Odontesthes bonariensis has a strong thermolabile
sex determination (TSD). The proportion of females
gradually varies from 100% at 15-19ºC to 0% at 29ºC
when the larvae are kept at different temperatures between
the 1-5th after hatching (Strüssmann et al., 1997).
These results make pejerrey an interesting model to study
the effects of the temperature on the mechanisms of sex
determination and differentiation in teleost species.
In this species, the ovarian differentiation was first
recognized at week 7 at 17ºC and at week 4 at 24ºC,
meanwhile testicular differentiation began at week 7 at 24ºC and at week 4 at 29ºC. Blood vessels can be first
observed at the gonads at week 6, 4, and 3 at 17, 24, and
29°C, respectively (Strüssmann et al., 2004). Thus, blood vessels are formed just before or concomitant with the
first signs of commitment to either sex at all temperatures.
These observations are consistent with the concept
of extragonadal regulation of sex differentiation in
this species.
During development, either FSH and LH, are expressed
in the anterior pituitary gland during the process
of gonadal determination/differentiation (Miranda et al., 2001a) and both GtHs show a clear correlation
with the increase or decrease of the number of GnRH
neurons located at the POA and the identification of
immunoreactive GnRH fibers in the pituitary gland
(Miranda et al., 2001a, 2003b). Also, the number of
FSH, LH and GnRH neurons at the POA varies according
to the incubation temperature suggesting a correlation
with TSD. Taking together, these results suggested
that the synthesis and release of GtHs may be regulated
by GnRH before or just at the moment of sex determination
showing that the hypothalamic-pituitary-gonadal
axis is active during the time of sex determination in
this species (Miranda et al., 2003; Strüssmann et al.,
2004). It can also be hypothesized that GtHs regulate
the production of sex steroids in the gonadal primordia
and/or in the interrenal gland during the sexual determination/
differentiation period (Strüssman et al., 2004).
Control of pejerrey reproduction in captivity
A series of studies were performed in order to optimize
pejerrey reproduction in captivity, including the
increase of sperm volume and the synchronization of
spawning by hormonal treatments.
In pejerrey, the sperm volume obtained by stripping
of males either in natural conditions or captivity is
very scarce (Strüssmann, 1989; Miranda et al., 2001b),
being necessary several males to fertilize the eggs produced
by a single female (Calvo et al., 1977).
In pejerrey males it was possible to increase sperm
volume by environmental and hormonal treatments practically
during all the year around without affecting the
sperm concentration and motility. These treatments included: increase of the light phase of photoperiod, human
chorionic gonadotropin (hCG), heterologous pituitary
extracts and GnRH superactive analogues (GnRHA)
injection (Miranda et al., 2001b; 2005). In order to
compare all hormonal treatments, an experiment using
the doses previously demonstrated to induce a 5 time
increase of expressible milt was performed. The treatment
with sGnRH-A was the most effective. However,
the use of hCG is recommended because of its effectiveness
to stimulate pejerrey spermiation in low doses,
its low cost and availability (Fig. 3).
FIGURE 3. Volume of expressible milt in Odontesthes bonariensis 24 hours after the administration of 0.7% saline solution (Control), hCG: 78 IU/kg (A), carp pituitary extracts: 10mg/kg (B), salmon pituitary extracts: 10 mg/kg (C), sGnRH-A: 5 mg/Kg (D) and mGnRH-A: 2.5 mg/kg (E). Values represent mean ± SEM, n=8 per treatment. Significant differences between treatments respect control group are indicated by different superscript letters (P< 0.05).
In the case of females, a commercial sustained release
system (Ovaplant, Syndel, Vancouver, Canada)
was used in order to synchronize spawning. Pejerrey
females (250 g) in late vitellogenesis with no signals of
final oocyte maturation, were intraperitoneally implanted
either with pellets having 75 mg of a sGnRH-A
or blank pellets. The first groups of eggs were obtained
after 60 hours in the sGnRH-A group and one week
later it was shown that 82% of sGnRH-A implanted females
had spawned meanwhile none of the control implanted
females spawned during that period.
Although much work is necessary in order to find
easier and non expensive ways to control pejerrey reproduction
in captivity, these results demonstrated that
is possible to use hormonal manipulation to induce reproduction
in this species. In this context, it is important
to think that even though the use of endocrine agents
can be use to induce reproduction in this species, the
study of environmental and maintenance conditions can
also be effective to synchronize reproduction in pejerrey.
Acknowledgements
This work was supported in part by grants of ANPCYT, Argentina (Pict 01-04424, Pict 01-12168 and Pict Redes 00528), Fundación Antorchas; CONICET PEI# 6439 to G.M.S and PEI#6438 to LAM and Ministry of Education, Culture, Sports, Science and Technology of Japan (#15201003) to Carlos Strüssmann. The authors also thank Jim Powell from Syndel International Inc for his generous donation of Ovaplant.
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Received on April 10, 2005.
Accepted on June 1, 2005.