Fish recombinant gonadotropins
by
Berta Levavi-Sivan (1), Matan GoLan (1), Joseph aizen (1) & abigail eLizur (2)
AbsTrAcT. - Pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play central
roles in regulating gametogenesis and the production of gonadal hormones, in fish as in other vertebrates. Pituitary gonado-
tropins are composed of two non-covalently associated polypeptide subunits, which must be glycosylated, folded, and
assembled as a heterodimer to be biologically active. Since biotechnology for the production of recombinant proteins has
been greatly advanced, it is possible that fish FSH and LH are produced biotechnologically as an alternative way for vari-
ous manipulations in modern aquaculture. This review discusses different platforms currently being applied for recombi-
nant piscine gonadotropin production.
Key words. - expression system - Yeast - insect cells - estradiol - Follicle - Stimulating hormone - Luteinizing hormone.
Cybium 2008, 32(2) suppl.: 17-21.
(1) Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University,
PO Box 12, Rehovot 76100, Israel. [sivan@agri.huji.ac.il]
(2) Faculty of Science, Health and Education, University of the Sunshine Coast, Maroochydore DC, Qld 4558, Australia.
Introduction
it has been almost a century since the laboratories of
Cushing and Aschner performed the first pituitary ablation
studies, implicating this gland as a primary regulator of ver-
tebrate reproduction. The gonadotropic effects of pituitary
extracts were soon associated with two distinct endocrine
factors, known as gonadotropins (GTHs) that became desig-
nated as follicle-stimulating hormone (FSH) and luteinizing
hormone (LH) (Campbell, 2005).
Phylogenetic analyses based on the amino acid sequenc-
es of vertebrate gonadotropin subunits, either derived from
chemically isolated peptides or deduced from cDnas,
revealed monophylogenetic lineage for each subunit. The
observed common ancestral origin for piscine and tetrapod
gonadotropin subunits has justified the use of the terms FSH
and LH for the fish GtH I and GtH II, respectively (Yaron et
al., 2003). In fishes, FSH is considered to regulate early
phases of gametogenesis, such as vitellogenesis and sperma-
togenesis, whereas LH is responsible for the final maturation
processes, such as oocyte maturation, ovulation, spermia-
tion, and milt production (Yaron et al., 2003).
The gonadotropins are all glycoproteins comprised of
dissimilar subunits designated α and β; the α subunit is the
same across hormones within a species, while the β subunits
are unique to each hormone (Pierce and Parsons, 1981).
Since GTHs are heterodimeric glycoproteins, the prokaryot-
ic expression systems (bacteria) that do not carry out post-
translational modifications of proteins are not appropriate
for expression of biologically active gonadotropins. Howev-
er, eukaryotic cell culture systems (amoeba, yeast, insect and
mammalian) have been established and small scale biologi-
cally active recombinant fish GTHs successfully produced in
these systems.
The biotechnology industry is currently experiencing an
extreme shortage of manufacturing capacity for recombinant
proteins. as a result, a growing number of biological systems
are being evaluated for the production of these valuable pro-
teins. Although some have been used for many years, others
are relatively new and still experimental. Factors such as
scale-up, total annual production, speed of production set-
up, post-translational modifications and regulatory issues
come into play in choosing the system that is most suitable
for any given target protein. This review discusses different
systems currently being applied for recombinant piscine
gonadotropin production.
Recombinant fish gonadotropin production platforms
Thus far, recombinant gonadotropins have been produced
from 10 fish species, representing four teleostean orders,
using various expression hosts: GPα of carp (Cyprinus car-
pio; Huang et al., 1991), FSH and LH of gilthead seabream
(Sparus aurata; Meiri et al., 2000), goldfish (Carassius
auratus; Kobayashi et al., 2003, Kobayashi et al., 2006)
Manchurian trout (Brachymystax lenok; Cui et al., 2007) and
channel catfish (Ictalurus punctatus; zmora et al., 2003) by
in vitro or in vivo insect cells; FSH and LH of African catfish
Clarias gariepinus by amoeba Dictyostelium discoideum
(vischer et al., 2003); FSH of Japanese eel (Anguilla japoni-
ca; Kamei et al., 2003) and FSH and LH of tilapia (Oreo-
chromis mossambicus; Kasuto and Levavi-Sivan, 2005,
aizen et al., 2007a, 2007b) by yeast; Manchurian trout (Choi
et al., 2005) and Zebrafish (Danio rerio; So et al., 2005) by
mammalian cells; and LH and FSH of goldfish by Rainbow
trout embryos (Morita et al., 2003,2004) (Tab. i).
Yield
Bacteria have proven useful as bioreactors because they
are grown easily at any scale. However, they are limited in
their ability to perform the post-translational protein modifi-
cations necessary for the gonadotropin biological activity.
Mammalian cells can perform complex post-translational
modifications, although the costs associated with scaling
these systems up for mass-production purposes are extremely
high. Certain eukaryotic systems, such as yeast and insects,
can be scaled-up with relative ease, are capable of post-trans-
lational modifications, and may also offer adequate produc-
tion yields, but they have unique glycosylation patterns,
which are not necessarily compatible with that of the native
protein/receptor. Transgenic fish have a potentially large pro-
duction capacity at lower costs than mammalian cell culture
but involve relatively slow and expensive production set-up
and have yet to cross many regulatory hurdles.
it is interesting to note that in the P. pastoris yeast sys-
tem, the yield of the gonadotropin heterodimer was signifi-
cantly lower than that of the monomer, probably because of
the complex structure of the former (Kasuto and Levavi-
Sivan, 2005, Aizen et al., 2007a). However, in the drosophila
cells the monomer was toxic to the cells and only the het-
erodimer could be produced at high levels (Zmora et al.,
2003).
Recombinant fish gonadotropins Le v a v i -Si v a n e t a l .
18 Cybium 2008, 32(2) suppl.
Table I. - Recombinant fish gonadotropins expressed at different platforms.
Structural integrity of recombinant gonadotropins
The molecular mass estimates of recombinant FSH and
LH vary considerably, however were in the range of those
reported for native teleost species; between 15 kDa and 28
kDa for the subunits and between 29 kDa and 55 kDa for the
intact hormones (Weltzien et al., 2004 and Tab. I). Because
the amino acid composition of individual subunits is similar
for of all teleosts species (Yaron et al., 2003) the different
molecular mass estimates may result from differences in the
degree of glycosylation.
Glycosylation of recombinant gonadotropins
among gonadotropin subunits, the sequences of the com-
mon α-subunit show the highest degree of conservation
across vertebrates. Sequence comparisons exhibited similar-
ities ranging from 60% to 90%, largely due to the carboxyl-
terminus half, which is the most conserved part of the mole-
Le v a v i -Si v a n e t a l . Recombinant fish gonadotropins
Cybium 2008, 32(2) suppl. 19
Table I. - Continued.
cule (Yaron et al., 2003). Fish FSHβ-subunits are more
divergent than the LHβ-subunits (average identities of 53%
and 67%, respectively) mainly due to a rapid change which
occurred during the evolution of teleosts in general, and that
of perciform species in particular.
The N-terminus of teleosts FSHβ sequences exhibits an
unexpected divergence at asparagine (N), putative glyco-
sylation sites, which are most conserved in FSHβ of other
vertebrates. The teleost GPα subunit, FSHβ subunit and
LHβ subunit carries two, one or two N-linked oligosaccha-
ride, respectively, and that consists of a heterogeneous array
of neutral, sulfated, and sialylated structures. The sugars,
amounting to more than 30% of the gonadotropin mass, form
the major chemical basis of the charge heterogeneity of GTH
isoforms, in the pituitary and bloodstream of mammals and
are essential for GTH intracellular folding, secretion, clear-
ance from blood, binding and signaling at the target-cell
level.
Deglycosylation of recombinant fish FSH and LH with
PnGase F, which hydrolyzes all types of n-glycan chains,
reduced the glycosylated forms to the nascent translated pro-
tein. This implies that in Japanese eel, tilapia, goldfish and
trout, the carbohydrate modifications on the mature protein
occur exclusively through N-linked, and not at all through
o-linked, glycosylation (Kamei et al., 2003, Kasuto and
Levavi-Sivan, 2005, Aizen et al., 2007a, Morita et al., 2004,
Ko et al., 2007). Deglycosylation experiments examining
other species of teleosts are needed in order to determine if
in all fish GTHs the glycosylation is exclusively through
N-linked or in some species it might have also O-linked oli-
gosaccharides. in contrast, mammalian glycoprotein hor-
mones carry both o-linked and n-linked oligosaccharides.
The role of glycosylation in the piscine gonadotropins is still
unclear and should be further elucidated.
Gene expression in response to application of recombinant
gonadotropins
The mRNA levels of Cyp19a1a and Cyp19a1b (the ovar-
ian and brain forms of aromatase) rose dramatically after
injecting recombinant grouper LH, concomitantly with
secretion of estradiol, while mRNA levels of GnRH dropped
and that of LHβ and GPα increased (Cui et al., 2007).
The gene expression of different enzymes along the ster-
oid biosynthesis pathway should be elucidated in several
other teleost groups, especially in fish bearing asynchronous
ovaries. Using recombinant gonadotropins which can be
administered together or separately, can aid in elucidating
the physiological events associated with secretion of each of
the different steroids.
Sex steroid production
all the recombinant LH and/or FSH, prepared by the dif-
ferent systems, increases the secretion rates of e2 and/or
androgen in females and males, respectively, when applied
in vitro to gonadal fragments (Tab. i), showing that the
recombinant proteins (with all the necessary modifications)
were able to activate the proper gonadotropin receptor.
Most of the species that their gonadotropins were studied
posses synchronous ovaries (catfish, eel, trout, and grouper)
meaning that their ovaries contain, at the same time, only
one generation of follicles, all at the same stage. We used
tilapia that posses asynchronous ovaries, thus their ovaries
contain several generations of follicles, at different stages, at
the same time. in order to elucidate the differential role of
each of the gonadotropins on the secretion of estradiol along
the reproductive cycle, we have exposed tilapia previtello-
genic ovarian fragments (GSI = 0.004% ± 0.0002%;
BW = 24.1 gr ± 2.23), to various doses of recombinant tFSH
or tLH according to (aizen et al., 2007a). As shown in figure
1 estradiol was increased in a dose dependent manner in
response to both gonadotropins, with a marked increase in
response to FSH. Both tilapia pituitary extract (TPe) and
hCG, a mammalian gonadotropin that is commonly used in
induced spawning of various fish species, were also potent
in the induction of E2 secretion (Fig. 1). We have recently
found that both GnrH-stimulated and basal secretion of
11-KT is inhibited after injection of anti-recombinant FSHβ,
implicating the involvement of FSH in 11-KT secretion in
tilapia (aizen et al., 2007a). Taken together these results are
in accord with the role of FSH in the regulation of early
phases of gametogenesis (Yaron et al., 2003).
ELISA using recombinant gonadotropins
recombinant tilapia gonadotropins were recently used to
develop specific and homologous competitive ELISAs for
measurements of FSH and LH in the plasma and pituitary of
the fish, using primary antibodies against rtLHβ or rtFSHβ,
respectively, rtLHβα or rtFSHβα for the standard curves,
Recombinant fish gonadotropins Le v a v i -Si v a n e t a l .
20 Cybium 2008, 32(2) suppl.
Figure 1. - Secretion of estradiol from previtellogenic tilapia ova-
ries (GSI = 0.004% ± 0.0002%) in response to various concentra-
tions of recombinant tilapia FSH or LH, in response to tilapia pitu-
itary extract (TPE), or human chorionic gonadotropin (hCG).
and rtLHβ or rtFSHβ for coating. The sensitivity of the assay
was 15.84 pg/ml for tLH and 0.24 pg/ml for tFSH measure-
ments in the plasma, whereas for the measurements in the
pituitary, the sensitivity was 2.43 ng/ml and 1.52 ng/ml for
tLH and tFSH, respectively. The standard curves for tFSH
and tLH paralleled those of serially diluted pituitary extracts
of other cichlids, as well as of serially diluted pituitary
extract of seabream, european seabass and hybrid bass
(aizen et al., 2007b).
Conclusion
The glycoprotein hormones are an interesting group of
molecules not only because of their importance in reproduc-
tion and overall physiology but also because of their unique
structural features. The mammalian gonadotropins have
proven to be useful models for understanding protein folding
and protein-protein interactions, as well as for studying the
role of carbohydrates in protein function. The availability of
recombinant fish FSH and LH in sufficient quantities should
enable us to better define their physiological role and pitui-
tary control as well as to further explore the usefulness of
fish as a model system for understanding gonadotropin func-
tion in vertebrates.
References
aizen J., KaSuTo H., GoLan,M., zaKaY H. & B. Levavi-
SIVAN, 2007a .- Expression and characterization of biologi-
cally active recombinant tilapia FSH: Immunohistochemistry,
stimulation by GnrH and effect on steroid secretion. Biol.
Reprod., 76: 692-700.
AIZEN J., KASUTO H. & B. LEVAVI-SIVAN, 2007b. - Develop-
ment of specific enzyme-linked immunosorbent assay for deter-
mining LH and FSH levels in tilapia, using recombinant gona-
dotropins. Gen Comp Endocrinol, 153: 323-332.
CAMPBELL R.K., 2005. - Molecular pharmacology of gonadotro-
pins. Endocrine, 26: 291-296.
CHOI E., KO H., SHIN J., KIM M.-A. & Y.C. SOHN, 2005. -
expression of gonadotropin genes in Manchurian trout Brachy-
mystax lenok and production of recombinant gonadotropins.
Fish. Sci., 71: 1193-1200.
CUI M., LI W., LIU W., YANG K., PANG Y. & L. HAORAN,
2007. - Production of recombinant orange-spotted grouper
(Epinephelus coioides) luteinizing hormone in insect cells by
the baculovirus expression system and its biological effect.
Biol. Reprod., 76, 74-84.
HUANG C., HUANG F., CHANG G., CHANG Y., LO C., FRAS-
er M. & T.B. Lo, 1991. - expression of two forms of carp
gonadotropin alpha subunit in insect cells by recombinant bac-
ulovirus. Proc. Natl. Acad. Sci. USA, 88(17): 7486-7490.
KAMEI H., OHIRA T., YOSHIURA Y., UCHIDA N., NAGASAWA
H. & K. AIDA, 2003. - Expression of a biologically active
recombinant follicle stimulating hormone of Japanese eel
Anguilla japonica using methylotropic yeast, Pichia pastoris.
Gen. Comp. Endocrinol., 134: 244-254.
KASUTO H. & B. LEVAVI-SIVAN, 2005. - Production of biologi-
cally active tethered tilapia LHβα by the methylotrophic yeast
Pichia pastoris. Gen. Comp. Endocrinol., 140: 222-232.
KO H., PARK W., KIM D.-J., KOBAYASHI M. & Y.C. SOHN,
2007. - Biological activities of recombinant Manchurian trout
FSH and LH: Their receptor specificity, steroidogenic and vitel-
logenic potencies. J. Mol. Endocrinol., 38: 99-111.
KOBAYASHI M., MORITA T., IKEGUCHI K., YOSHIZAKI G.,
SUZUKI T. & S. WATABE, 2003. - Production of recombinant
goldfish gonadotropins by baculovirus in silkworm larvae. Fish
Physiol. Biochem., 28: 469-471.
KOBAYASHI M., MORITA T., IKEGUCHI K., YOSHIZAKI G.,
SUZUKI T. & S.WATABE, 2006. - In vivo biological activity
of recombinant goldfish gonadotropins produced by baculovi-
rus in silkwonn larvae. Aquaculture, 256: 433-442.
MEIRI I., ZMORA N. & A. ELIZUR, 2000. - Functional expres-
sion of recombinant seabream FSH and LH in baculovirus
infected insect cells. In: Sixth International Symposium on
Reproductive Physiology of Fish. (Norberg B. et al., eds), p.
488. Univ. of Bergen.
MORITA T., YOSHIZAKI G., KOBAYASHI M. & T. TAKEUCHI,
2003. - Production of biologically-active recombinant goldfish
gonadotropins in transgenic rainbow trout. Fish Physiol. Bio-
chem., 28: 473-474.
MoriTa T., YoSHizaKi G., KoBaYaSHi M., WaTaBe S. & T.
TAKEUCHI, 2004. - Fish eggs as bioreactors: the production
of bioactive luteinizing hormone in transgenic trout embryos.
Transgenic Res., 13: 551-557.
PIERCE J.G. &T.F. PARSONS, 1981. - Glycoprotein hormones:
structure and function. Annu. Rev. Biochem., 50: 465-495.
SO W.-K., KWOK H.-F. & W. GE, 2005. - Zebrafish gonadotropins
and their receptors: II. Cloning and characterization of zebrafish
follicle-stimulating hormone and luteinizing hormone subunits
their spatial temporal expression patterns and receptor specifi-
city. Biol. Reprod., 72: 1382-1396.
VISCHER H.F., GRANNEMAN J.C.M., LINSKENS M.H.K.,
SCHULZ R.W. & J. BOGERD, 2003. - Both recombinant Afri-
can catfish LH and FSH are able to activate the African catfish
FSH receptor. J. Mol. Endocrinol., 31: 133-140.
WELTZIEN F.A., ANDERSSON E., ANDERSEN O., SHALCHI-
AN-TABRIZI K. & B. NORBEG, 2004. - The brain-pituitary-
gonad axis in male teleosts, with special emphasis on flatfish
(pleuronectiformes). Comp. Biochem. Physiol. A, 137:
447-477.
Yaron z., Gur G., MeLaMeD P., roSenFeLD H., eLizur
A. & B. LEVAVI-SIVAN, 2003.- Regulation of fish gonadotro-
pins. Int. Revr. Cytol., 225: 131-185.
ZMORA N., KUMAR S., KAZETO Y. & J.M. TRANT, 2003. -
Production of channel catfish (Ictalurus punctatus) recom-
binant gonadotropins using the S2 Drosophila cell line system.
Fish Physiol. Biochem., 28: 475-477.
Le v a v i -Si v a n e t a l . Recombinant fish gonadotropins
Cybium 2008, 32(2) suppl. 21