Gonadotropin-Releasing Hormone (GNRH) and Its Natural Analog

Review
Gonadotropin-releasing hormone (GnRH) and its natural

analogues: A review
Falk Schneider
a,
*
, Wolfgang Tomek
a
, Carsten Grundker
b
a
Department of Reproductive Biology, Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2,
D-18196 Dummerstorf, Germany
b
Department of Gynecology and Obstetrics, Georg-August-University, Gottingen, Germany
Received 28 July 2005; received in revised form 20 January 2006; accepted 17 March 2006
Abstract
The pivotal role of gonadotropin-releasing hormone (GnRH) during the hormonal regulation of reproductive processes is
indisputable. Likewise, many factors are known to affect reproductive function by inuencing either GnRH release from
hypothalamus or pituitary gland responsiveness to GnRH. In veterinary medicine, GnRH and its agonists (GnRHa) are widely
used to overcome reduced fertility by ovarian dysfunction, to induce ovulation, and to improve conception rate. GnRHa are,
moreover, integrative part of other pro-fertility treatments, e.g. for synchronization of the estrous cycle or stimulation for embryo
transfer. Additionally, continuous GnRH which shows desensitizing effects of the pituitary-ovarian axis has been recommended for
implementation in anti-fertility treatments like inhibition of ovulation or reversible blockade of the estrous cycle. Just as much,
another group of GnRH analogues, antagonists, are now in principle disposable for use.
For a few decades, GnRH was thought to be a unique structure with a primary role in regulation gonadotropins. However, it
became apparent that other homologous ligands of the GnRH receptor (GnRHR) exist. In the meantime, more than 20 natural
variants of the mammalian GnRH have been identied in different species which may compete for binding and/or have their own
receptors. These GnRH forms (GnRHs) have apparently common and divergent functions. More studies on GnRHs should
contribute to a better understanding of reproductive processes in mammals and interactions between reproduction and other
physiological functions. Increased information on GnRHs might raise expectations in the application of these peptides in veterinary
practice. It is the aimof this reviewto discuss latest results fromevolutionarily based studies as well as rst experimental tests and to
answer the question howrealistic might be the efforts to develop effective and animal friendly practical applications for endogenous
GnRHs and synthetic analogues.
# 2006 Elsevier Inc. All rights reserved.
Keywords: Reproduction; Farm animals; GnRH; Hypothalamus; Gonadotropin
Contents
1. Introduction to gonadotropin-releasing hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 692
2. Biosynthesis and storage of GnRH-I and its natural analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 692
3. Release mechanisms of GnRH-I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 694
4. Reception of GnRH-I and its variants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 696
www.journals.elsevierhealth.com/periodicals/the
Theriogenology 66 (2006) 691709
* Corresponding author. Tel.: +49 38208 68757; fax: +49 38208 68752.
E-mail address: falk.schneider@fbn-dummerstorf.de (F. Schneider).
0093-691X/$ see front matter # 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2006.03.025
5. Hypophyseal and extrahypophyseal effects of natural GnRHs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698
6. Use of GnRH-I and its natural forms in farm animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 700
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 703
1. Introduction to gonadotropin-releasing
hormone
GnRHbelongs to a group of neuropeptides originally
discovered and successfully isolated as factors of
hypothalamic origin that control secretions of the
anterior pituitary gland. GnRH inuences reproductive
processes, mainly by regulating pituitary gonadotropin
synthesis and release, which in turn modulate ster-
oidogenesis and gametogenesis. This pivotal role may
explain why the interest in GnRH is still going on since
its primary structure was revealed in pigs [1] and sheep
[2]. GnRH is expressed, apart from hypothalamus, in
numerous peripheral tissues including gonads and
placenta [310]. Hypothalamic and extrahypothalamic
GnRH are known as integrated part of multiple
paracrine/autocrine axes. Hypothalamic GnRH, how-
ever, is in the focus of attention of the present review.
There are currently 23 identied naturally occurring
GnRH analogues (GnRHs) across the vertebrate species
[1113]. These variants show multiple substitutions in
their amino acid (AA) sequence when compared with the
mammalian GnRH (mGnRH), and they were widely
distributed in tissues suggesting that they have acquired
signicant functions throughthe phylogeny. GnRHs have
been explored during last years in various classes of
vertebrates, mainly in shes and mammals, but also in
protochordates, that are phylogenetically distant from
mammalian vertebrates. The species-related designation
of single GnRH isoforms may be difcult to survey and
confusing under circumstances. In this review mGnRH
will always be designated, according to [14], GnRH-I,
chicken GnRH-II (cGnRH-II) GnRH-II, and salmon
GnRH (sGnRH) GnRH-III. Other GnRHs will be
preferably discussed by citing the traditional names
which have been introduced in the literature. It was
assumed that dogsh GnRH, GnRH-II, and GnRH-III are
ancient forms which could be likely found in inverte-
brates [15]. At this stage, the exploration of the primary
structure of GnRHs has been completed only in tunicates
and octopus. More information seems to be necessary to
complete the phylogenetic tree of GnRH and to
characterize the ontogenic development of single GnRHs
in different species [16].
GnRH-I and its variants are thought to have
neuroendocrine, as well as neurotransmitter and neuro-
modulatory functions [13,17]. The neuroendocrine axis
plays the central role in the control of reproduction, and
GnRH-I is well known to integrate internal and external
signals of the nervous system to reproductive system
[15]. In invertebrates, a similar role of GnRHs may have
evolved resulting in systems that assure the maintenance
of species under changing environments. However,
GnRH-I also acts as a neuromodulator probably by
down-regulation of its neighboring genes for particular
protein-tyrosin phosphatases [18]. This GnRH-I function
could be an important link of the common molecular
mechanism underlying the diverse functions of GnRHs.
It is evident that at least two of these natural GnRHs
are present in more than 80 vertebrate species [12
14,1921]. A better understanding is, however, still
needed to evaluate the specic contribution of GnRHs
to the control of mammalian reproduction by GnRH-I in
mammals. The practical usefulness of GnRH-I and its
synthetic analogues (agonists and antagonists) has
achieved a high level. It is the purpose of the present
review to discuss in this context the prospects for
GnRH-II and other GnRHs although the exact functions
of these peptides are yet to be dened [22].
2. Biosynthesis and storage of GnRH-I and its
natural analogues
Like many other proteins and peptides, GnRH-I and
its analogues are enzymatically processed from larger
precursors (Fig. 1). The complementary DNA consists
of the decapeptide, extended at the amino (N) terminus
by a signal peptide, and at the carboxy (C) terminus by a
Gly-Lys-Arg sequence followed by the GnRH-asso-
ciated peptide (GAP).
In the proGnRH-I, the GAP sequence is 56 AA long
but it shows, like the signal peptide, considerable
variations between several mammals [3] like that of
pro-GnRHs of other species [23,24]. GAP which is
F. Schneider et al. / Theriogenology 66 (2006) 691709 692
Fig. 1. Schematic representation of the prepro-GnRH precursor pro-
teins. The 5
0
-untranslated region (5
0
utr) is followed by the signal
peptide (SP), the GnRH, processing signal (*), the GnRH-associated
peptide (GAP), and the 3
0
-untranslated region (3
0
utr). Adapted to
Sherwood et al. [3].
consequently co-secreted with GnRH [10,25] may be
involved in the correct processing and packing of the
decapeptide [3]. The processing of GnRH-I occurs
within about 1000 GnRH neurons that do not reside in
dened cytoarchitectonic bound-areas among different
species [26]. GnRH neurons are distributed in a loose
array along the ventral medial forebrain from the
posterior olfactory bulbs to the arcuate nucleus. GnRH
neurons are found in the vicinity of the olfactory
placode during prenatal development, and then the
GnRH-producing cells migrate through the nasal
system into the forebrain [27]. Therefore, differences
in GnRH-producing areas within the brain appear to
result from greater or lesser penetration along the
olfactory-forebrain-hypothalamus continuum to the
median eminence. The unusual developmental course
of GnRH neurons may represent a vestige of the
pheromonal control of reproductive function and, thus,
explain the release and transportation of GnRH into the
hypothalamus-pituitary portal vasculature and the
clinical link between reproductive failure and olfactory
dysfunction, respectively [28].
Apart from GnRH-I and, very often, GnRH-II
[17,2931] the presence of a third form, lamprey
GnRH-III (lGnRH-III), has been determined in neurons
of the preoptic-hypothalamic region of the rat, both
alone and together with GnRH-I [32]. Others were,
however, not able to isolate lGnRH-III in humans [8,30]
or in hamster and rat brains [29]. The primary AA
structure of most GnRHpeptides is common: a length of
10 AA and conserved AA in positions 1 to 4, 9, and 10
(Table 1). The compositions of the peptide chains of
natural GnRHs show a range of substituted AA from
one (e.g. cGnRH-I) to ve (lGnRH-I) compared with
GnRH-I. The AA sequence of GnRH-II shows, for
example, differences in positions 5 (His for Tyr), 7 (Trp
for Leu), and 8 (Tyr for Arg), compared with that of
GnRH-I. There are, however, some striking exceptions
of common composition, e.g. the sequences of guinea
pig (gp)GnRH, lGnRH-I, and octopus (o)GnRH [15,17]
(Table 1).
The listing of GnRHs demonstrates that the main
part of them was found in evolutionarily old life forms
and fewer variants occur in mammals, respectively [33].
Table 1
Amino acid composition of GnRH-I and its 23 natural analogues in vertebrates and invertebrates
Amino acid
1 2 3 4 5 6 7 8 9 10
Vertebrate
Mammal (GnRH-I) pGlu His Trp Ser Tyr Gly Leu Arg Pro Gly.NH2
Guinea Pig pGlu Tyr Trp Ser Tyr Gly Val Arg Pro Gly.NH2
Chicken-I pGlu His Trp Ser Tyr Gly Leu Gln Pro Gly.NH2
Rana pGlu His Trp Ser Tyr Gly Leu Trp Pro Gly.NH2
Seabream pGlu His Trp Ser Tyr Gly Leu Ser Pro Gly.NH2
Salmon (GnRH-II) pGlu His Trp Ser Tyr Gly Trp Leu Pro Gly.NH2
Whitesh pGlu His Trp Ser Tyr Gly Met Asn Pro Gly.NH2
Medaka pGlu His Trp Ser Phe Gly Leu Ser Pro Gly.NH2
Catsh pGlu His Trp Ser His Gly Leu Asn Pro Gly.NH2
Herring pGlu His Trp Ser His Gly Leu Ser Pro Gly.NH2
Chicken-II (GnRH-II) pGlu His Trp Ser His Gly Trp Tyr Pro Gly.NH2
Dogsh pGlu His Trp Ser His Gly Trp Leu Pro Gly.NH2
Lamprey-III pGlu His Trp Ser His Asp Trp Lys Pro Gly.NH2
Lamprey-I pGlu His Tyr Ser Leu Glu Trp Lys Pro Gly.NH2
Invertebrate
Octopus pGlu Asn Tyr His Phe Ser Asn Gly Trp His Pro Gly.NH2
Tunicate-I pGlu His Trp Ser Asp Tyr Phe Lys Pro Gly.NH2
Tunicate-II pGlu His Trp Ser Leu Cys His Ala Pro Gly.NH2
Tunicate-III pGlu His Trp Ser Tyr Glu Phe Met Pro Gly.NH2
Tunicate-IV pGlu His Trp Ser Asn Gln Leu Thr Pro Gly.NH2
Tunicate-V pGlu His Trp Ser Tyr Glu Tyr Met Pro Gly.NH2
Tunicate-VI pGlu His Trp Ser Lys Gly Tyr Ser Pro Gly.NH2
Tunicate-VII pGlu His Trp Ser Tyr Ala Leu Ser Pro Gly.NH2
Tunicate-VIII pGlu His Trp Ser Leu Ala Leu Ser Pro Gly.NH2
Tunicate-IX pGlu His Trp Ser Asn Lys Leu Ala Pro Gly.NH2
Bold letters: primary structure of GnRH-I and identical amino acids in variants. Adapted to Gorbman and Sower [15] and Millar et al. [17].
The most ancient GnRH form is thought to be oGnRH
[34]. Another form of GnRH, 9-hydroxyproline GnRH-
I (Hyp
9
-GnRH), has been reported to be present in
various mammals like rats and dogs. The peptide has a
nearly identical structure to GnRH-I but the small
difference in the 9th AA makes it less sensitive to prolyl
endopeptidase [3537]. The answer to the question
whether various GnRH forms [38] and their genes
[21,3943], respectively, are present in a distinct
species may have some importance for the under-
standing of the evolution and be helpful to draw up
specic functions of GnRHs in mammals. The
phylogenetic tree which was recently established [15]
contains the AA structures of the natural GnRHs
(Fig. 2).
The GnRH-II structure ([His
5
,Trp
7
,Tyr
8
]-GnRH;
Table 1) has been conserved over 500 million years
[12,21]. GnRH-II is one of the GnRHs that appears to be
predominantly expressed in other tissues such as the
reproductive system [22]. In the brain of mammals, the
vast majority of GnRH-II cell bodies are localized to the
midbrain, with a few present in hypothalamic and
extrahypothalamic regions [44] and a minority of ber
projections fromneurons containing GnRH-II project to
the median eminence or regions that regulate gonado-
tropin release [45]. In the human, however, GnRH-II is
expressed at signicantly higher levels outside the
brain, e.g. in the kidney, bone marrow, and prostate [40].
Other species-specic prerequisites of the biosynthesis
of GnRHs were reported for lGnRH-III [46] and GnRH-
III [47] in several mammals.
3. Release mechanisms of GnRH-I
The release of hypothalamic GnRH-I is not
continuous but pulsatile. The episodic release of GnRH
during the female reproductive cycle is seen as a
prerequisite for fertility. Multiple rhythms interact to
generate GnRH-I pulses, thus high-frequency burst
rhythms decrease during peaks in the low-frequency
rhythm with approximately 20 min duration. The
pulsatile release pattern is the predominant one in
most physiological circumstances. The sole exception
to pulsatility is at the time of the preovulatory LH.
Several theses were postulated to understand the
mechanisms underlying episodic GnRH-I release.
One of the theses is that interactions among GnRH
neurons should be crucial for synchronizing and
modulating the low-frequency rhythm which is asso-
ciated with hormone release [48].
Numerous loops, ultra-short, short and long in
length, form a link between the hormone-secreting
tissues. Paracrine interactions among GnRH neurons
are known, for example, as the rst proposed ultra-short
loop [49]. The real frequency of GnRH pulses in an
individual is species-specic and depends on its gonadal
hormone status and numerous other factors [50,51]. The
role of the ovarian steroids estradiol (E
2
) and
progesterone (P
4
) in the control pulsatile GnRH
secretion and generation of the preovulatory GnRH
surge to induce the release of the LH surge seems to be
well investigated [23,52]. Both steroids interfere with
gonadotropin regulation by modifying the secretion of
Fig. 2. Phylogenetic analysis of 24 GnRH primary amino acid structures (bold letters: GnRHs with demonstrated or supposed importance to
mammalian reproduction). Adapted to Gorbman and Sower [15].
GnRH or exhibit a direct effect on gonadotropin-
secreting cells in the anterior pituitary gland. There is
some evidence to suggest that the negative feedback
actions of E
2
are manifest in the suppression of GnRH
pulse amplitude and P
4
may primarily exert its effects
on GnRHpulse frequency. In the mid-luteal phase of the
estrous cycle, for example, the negative feedback of P
4
suppressed GnRH release as well as decreased the
expression of the GnRH mRNA in heifers [53]. In
sheep, the negative feedback of E
2
on the episodic
GnRH secretion was found to be highly effective during
anestrus [54]. The understanding of the effects of
gonadal steroids on synthesis of GnRH-I is less clear
than that on secretion [23]. Apart from direct effects
other effects are transmitted through steroid-responsive
neuronal systems in various parts of the brain. They can
override direct effects under special situations, e.g.
undernutrition and stress.
High levels of estrogens produced during the
preovulatory period exert a positive feedback action,
probably by culminating in the release of increased
GnRHamounts and gonadotropin surges. Previously, it
was assumed that E
2
stimulates the expression of the
gene encoding the GnRH receptor. The increase
precedes the preovulatory rise in circulating concen-
trations of E
2
. The enhanced pituitary sensitivity to
GnRH may occur as a result of a decrease in
concentrations of P
4
rather than in an increase in E
2
concentrations [55].
Moreover, there have been reported effects of
various hormones of non-reproductive organs and
tissues, e.g. cortisol from adrenals or leptin from fat
cells, which may act on the hypothalamus to inuence
pulsatile GnRH release and/or on the pituitary gland to
inhibit gonadotropin responsiveness to GnRH [5662].
The secretion of GnRH during the reproductive cycle is,
furthermore, the result of qualitative and quantitative
modications in neurotransmitter systems, e.g. cate-
cholaminergic, opioidergic and GABAergic systems,
which show great differences in their impact [8,23,63
70]. It was summarized [71] that many compounds can
potentially modify the GnRH-I-induced secretion of
gonadotropins by different ways:
Peptides, e.g. oxytocin, are transported from the
hypothalamus and directly acts on the gonadotrope.
There is an interaction of the peptides, e.g. neuropep-
tide Y, from the hypothalamus with GnRH-I.
Peptide elicits release of another substance that acts
on the gonadotrope and so produces a paracrine
mechanism of action, e.g. PACAP stimulates IL-6
release from folliculo-stellate cells.
Peptide is released froma cell within the pituitary, e.g.
galanin from lactotropes.
An autocrine process utilizing peptide synthesized
within the gonadotrope, e.g. substance P.
The gonadotropins LH and FSH are dimeric
glycoprotein hormones, showing a common a-subunit
while possessing distinct b-subunits, the latter con-
ferring biological actions [72]. The subunits are
encoded by different genes, and the expression may
be regulated in either a coordinate or differential fashion
[73]. Gonadal steroid and peptide hormones can exert
selective actions on one or more of the subunit gene
products [52,74]. It is a striking feature that GnRH
induces potentiation of its own action on the LH
response by the phenomenon self-priming which
means a biphasic LH response. Recently, it was argued
that ovarian factor(s) like the putative gonadotropin-
surge inhibiting factor/attenuating factor exerts inhibi-
tory effects on the amount of circulating gonadotropins
at the pituitary but rather at the hypothalamus level
itself. The steep increase in LH responsiveness occurs
only after the elimination of this inhibitory action by
GnRH [75]. Gonadal peptides may also be responsible
for the fact that the GnRH surge consistently continued
well beyond the surge of LH [76].
The importance of the episodic GnRH signal to the
pituitary gonadotropes was demonstrated over two
decades ago by Knobil and his colleagues. These studies
revealed that in the presence of a continuous GnRH
signal an episodic pituitary release of LH and FSH was
down-regulated to levels not compatible with fertility.
The continuous treatment by GnRH-I or GnRH-I
agonists was able, for example, to induce down-
regulatory effects in bovine pituitary gonadotrophs [77
81]. The examination of mechanisms of LH and FSH
biosynthesis revealed that the continuous treatment with
a GnRH-I agonist might inuence gonadotropin
biosynthesis by a post-transcriptional mechanism
because the contents of both hormones were suppressed
to a greater degree than their beta-subunit mRNAs [82].
Recent studies in which the release of FSHand LHwere
examined in pituitary portal or sinus cavernosus blood
samples have shown that, while there is a high degree of
synchrony between the pulsatile release of GnRH-I and
LH, FSH release is only associated with a small portion
of the GnRH pulses [13].
There are many experimental results which indicate
a different release pattern of LH and FSH during the
estrous cycle of untreated animals but the underlying
mechanisms are still unclear [52]. The existence of one
mechanism for the different regulation of LH and FSH
by a single neuroendocrine factor seems to be contra-
dictory. High-frequency GnRH pulses favour LH
release, whereas low-frequency pulses favour FSH
release. This discrepancy was the reason to assume that
an additional peptide exists which releases specically
FSH [83]. Among the natural GnRHs, both GnRH-II
[84,85] and lGnRH-III [86] or a closely related peptide
[46,87] have been suggested to be the putative FSH-RH
but there is neither a study conrming the presence of
GnRH-II in the pituitary portal blood [88] nor a
consensus over the functioning of lGnRH-III as FSH-
RH in mammals [11]. By applying a specic GnRH-I
antagonist in two sheep models, another group [89] was
able to demonstrate a constitutive component of GnRH-
I-independent basal LH release after termination of
pulses. But, more interestingly, a rst evidence was
provided by the authors that an acute GnRH-I-
independent component of episodic FSH secretion
exists which may, probably be triggered by a selective
GnRH-II effect or by a hypothalamic compound still
unknown. In other studies [13,52], where a different
packaging of both gonadotropins in storage granules
within the gonadotrope cells was detected, constitutive
pathways of FSH and basal LH release as well as a
GnRH-regulated pathway of pulsatile LH release were
also explored.
The distribution of GnRH-I and its natural analogues
in both the same and different neurons and brain areas
may lead to the conclusion that there is a common or a
sequential release of related peptides. The distinguished
feature of GnRH-II is, for example, its wide distribution
in extrahypothalamic regions of the brain compared
with GnRH-I [21]. The more the interest is growing to
analyze the specic release patterns and to discover the
behavioral roles of GnRHs the more a need is arising to
develop suitable models [22]. Models may, for example,
involve animals that are energetically challenged by
food restriction. Such animals showed increases in the
number of GnRH-II immunoreactive cell bodies and
ber dense area followed by a fast emptying of stored
peptide plus showing behavioral activity after refeeding
[31].
4. Reception of GnRH-I and its variants
Hypothalamic GnRH-I regulates the synthesis and
secretion of LH and FSH on pituitary gonadotrophs
through its receptor [90]. The density of the type-I
GnRHR on gonadotrophs is changing during various
stages of the estrous cycle [91]. Receptor concentrations
have been reported to increase up to day 18 of the
bovine estrous cycle [92] stimulated mainly by GnRH-I
itself and E
2
. Developing ovarian follicles produce
increasingly E
2
that substitutes the GnRH effect and
maintains high abundance of GnRHR mRNA in the
preovulatory period. It was discussed that P
4
exerts a
suppressive inuence [91,93,94], therefore, maximal
GnRHR expression and maximal sensitivity of gona-
dotrophs to GnRH is probably triggered by decreased
circulating concentrations of P
4
at luteolysis [95]. The
presence of a P
4
-responsive element in the 5
0
-anking
region of the human GnRHR suggests, however, the
regulation of this gene by P
4
. As explored recently, the
physiological effects of P
4
are mediated through two
receptor forms, the truncated form PR-A and PR-B.
Overexpression of PR-A was reported to decrease
GnRHR promoter activity both in pituitary and
placental cells but that of PR-B was found to exhibit
a cell-dependent transcriptional activity, e.g. to decrease
it only in pituitary cells [96].
From 1992/1993, since the initial isolation of the
cDNAs encoding the type-I GnRHR of the mouse
[97,98], rat [99,100], sheep [101,102], human
[103,104], cattle [105], pig [106] and, following this,
other species, a lot of studies were performed to
describe the molecular structure and organization of the
GnRHR gene. Mammalian pituitary type-I GnRHRs
share often over 80% AA identity. The cDNA of the
type-I GnRHR encodes, for example, a 327 AA protein
in mouse and rat, and a Lys residue more is present in
sheep and human [10]. Non-mammalian receptors show
not more than 4247% identity to those of mammals
and 5867% among each other, respectively [17].
The type-I GnRHR is a member of the G-protein-
coupled receptor (GPCR) family which is characterized
by an extracellular N terminus and an intracellular C
terminus linked by seven transmembrane (7TM; TM1
TM7) helices. The helices are connected by each three
extracellular and intracellular loops [91,107,108].
GPCRs comprise a large superfamily of proteins; more
than 1000 different members of that are still known
[109]. Receptor binding activates two specic GTP-
binding proteins (Gq/G11), which stimulate increased
phosphoinositol turnover by activating phospholipase C
(PLC). This enzyme leads to the generation of several
second messengers [110]. Among these, diacylglycerol
(DG) and inositol 1,4,5-tris-phosphate (IP3) are
critically important. DG leads to activation of protein
kinase C (PKC), and IP3 releases Ca
2+
from intracel-
lular pools [74]. Both events result in gonadotropin
synthesis, which is realized across the stimulation of the
steroidogenic factor 1 (SF-1) [111,112], followed by
secretion of LH and FSH. It has been demonstrated that
the GnRH signal transduction pathway involves an
increase in cAMP and an activation of members of the
mitogen-activated protein kinase (MAPK) family
[74,110]. To help understanding the molecular mechan-
isms involved in transcriptional regulation of the receptor
gene, the 5
0
ankingregionof the type-I GnRHRhas been
isolated, and there were explored several DNA regions
which are responsible for both basal expression and
GnRH-I-mediated homologous regulation of this gene in
pituitary cells [113]. The type-I GnRHR has a relatively
short third intracellular loop, and it lacks a Cterminal tail
[110]. Because the tail is unique among GPCRs, its loss
during evolution should be discussed. The structural
features described may be responsible for the relative
resistance of the type-I GnRHR to rapid homologous
receptor desensitization and slow internalization by
GnRH-I agonist-induced phosphorylation or binding b-
arrestin by sustained stimulation [114].
However, the presence of more than one forms of
GnRH in the same species implies the co-evolution of
cognate receptors. In all, three GnRH peptides and three
cognate receptors have been yet identied with distinct
distributions and functions [17]. Two or three different
types of GnRHR have been indeed identied in non-
mammalian species like sh and frog [20], but also in
Macaca mulatta [115,116]. In contrast, only one
functional GnRHR gene, the type-I GnRH gene, has
been identied in humans. Some ndings suggested that
a type-II GnRHR gene has arisen during vertebrate
evolution, is also present but vestigial in the human due to
the transcription of the opposite DNA strand to antisense
RNA [117]. The gene encoding the type-II GnRHR is,
however, functional in chimpanzee but partially or
completely silenced in a range of other mammals [33].
Antisera to one domain of the human type-II receptor, for
example, revealed immunoreactivity in pituitary and
brain cells of the human, rhesus monkey, sheep and
mouse [21]. The primate type-II GnRHR AA sequences
share 60% identity with amphibian type-II GnRHRs and
40% identity with the human type-I GnRHR [88]. That
means that the structure of monkey type-II GnRHR
resembles more closely non-mammalian types of
GnRHRs than mammalian type-I GnRHRs [20]. Like-
wise, non-mammalian GnRHRs contain also a C-
terminal cytoplasmatic tail, and both groups of receptors
have conserved Asp
2.50
/Asp
7.49
residues, while mamma-
lian GnRHRs contain Asn
2.50
/Asp
7.49
at these positions
[118]. It was shown by mutation products of the mouse
type-II receptor that Asp
7.49
in TM7 is an essential
component of ligand-mediated receptor activation and
the TM2 Asn
2.50
is essential for conguring and
expression of the receptor [17]. Finally, non-mammalian
and monkey type-II GnRHRs are more sensitive to
GnRH-II than GnRH-I [30,119]. Type-II receptors are
highly selective for GnRH-II, e.g. GnRH-II is approxi-
mately 400-fold more potent at type-II GnRHR than
type-I receptors [17] and has approximately 10% of
the activity of GnRH-I at the type-I receptor [30],
respectively.
In addition to the gonadotropes, type-I GnRHRs are
also expressed in many other tissues, e.g. breast, gonads,
the central nervous system, and some neoplastic tissues
[6,114,120123]. The presence of GnRH-I and type-I
receptors in the gonads of various vertebrate species,
including mammals, may reect a direct regulation of the
gonads as an early evolved function, and the neuroendo-
crine role in regulating the pituitary as a later
evolutionary development [11]. However, unlike the
hypothalamus and pituitary, the regulation of GnRH-I
and its receptor in the ovary is poorly understood [12]. In
human placenta, where GnRH-I is thought to playroles in
both growth and function, the expression of type-I
receptors is regulated by an autocrine mechanism [124].
Recently, it was shown, that GnRH-I and GnRH-II are
coexpressed in the rst-trimester decidua, suggesting
biological effects within and between maternal and fetal
cellular compartments [125]. As mentioned before, the
type-II GnRHR has been cloned from various vertebrate
species, including New and Old World primates. The
human gene homologue of this receptor is considered as
vestigial receptor, as reported [117], is frame-shifted and
has a different stop codon, and it appears that GnRH-II
signalling occurs through the type-I receptor [17]. A
growing number of extrapituitary GnRH-II actions in
humans have been demonstrated although a full-length
type-II receptor transcript has not yet been identied
[115]. Likewise, it is absent from chimpanzee, cow,
horse, sheep, rat and mouse in spite of its presence in
other species, e.g. pig[11]. This challenges the questionif
there is expressed a functional type-II receptor protein
expression in cells lacking the type-I receptor. It was
shown that the antiproliferative effect of GnRH-II on
human cancer cells is not mediated through the type-I
receptor like that of a GnRH antagonist [126]. The data
are in accord with a report from[127] demonstrating that
the human type-II GnRHR is functional, and its splice
variant determines the direction of the cellular response
to GnRH stimulation. More recently, an immunohisto-
logical study using a polyclonal antiserum against the
putative human GnRH-II receptor demonstrated the
existence of a GnRH-II receptor-like protein in human
placenta and in cancers of human reproductive organs
[128].
The sequences of the pig and monkey type-II
GnRHRs are 91% identical as well as their functional
properties, e.g. in terms of stimulating the IP3 production
by GnRH-I and GnRH-II, respectively [30]. Interest-
ingly, further variants of the type-II GnRHR exist in
sheepandcattlewhich donot appear to encode functional
7TM GPCRs. Similar observations were made in human
and pigs where receptor forms have been explored which
showed a shortened structure (Fig. 3) [30,88,129,130].
5. Hypophyseal and extrahypophyseal effects of
natural GnRHs
In invertebrates, which have no pituitary gland, the
diversity of anatomical distribution patterns observed for
GnRH or GnRH-like factors suggests that GnRHs may
have multiple functions. One of them could be related to
stimulation of reproduction by a direct action on the
gonads [11,15,131]. Such functions explored for GnRH
variants except GnRH-I in vertebrates would be
evolutionarily old whereas the evolutionarily younger
GnRH-I became responsive for specic tasks within the
HPGaxis. The questions are if, inhigher vertebrates, both
GnRH-I exerts functions other than stimulation of
pituitary and if GnRHs exert functions included
stimulationof gonadotropinrelease. Now, it is commonly
accepted that GnRH-I is integrated in some important
functions which fall into three categories [130]:
gonadotropin secretion;
sexual behavior;
regulation of peripheral reproductive tissues.
For a few decades, hypothalamic GnRH-I has been
assumed to be sufcient to control synthesis and release
of gonadotropins and sexual behavior, hence reproduc-
tive function. Furthermore, it was suggested that the
interactions of GnRH-I with ovarian steroids at the
pituitary level are responsible for the nal regulation of
LH and FSH release. As reviewed above, an increasing
number of peptides have been observed, however, to
affect gonadotropin activity during the following
decades. None of these factors has gained an
importance equal to that of GnRH-I. All data available
suggest that differential synthesis and release of
gonadotropins is caused by an altered pattern of
pulsatile GnRH-I secretion and triggered by activation
of pituitary type-I GnRHR [74].
Endogenous sources for GnRH and GnRH-like
factors were found in manifold tissues. The fast
destruction of the releasing hormone of hypothalamic
origin by specic peptidases might lessen peripheral
effects of them. However, GnRH-I inuences the
functions of other organs, such as the breast, placenta,
and ovary, in addition to its actions in the brain and
pituitary. Extrapituitary actions of GnRH-I which have
been manifold documented suggest that there is a
functioning GnRH/GnRHR system in these organs.
There is evidence for a paracrine/autocrine role of
GnRH-I in the regulation of ovarian processes like
follicular atresia and luteinization [132134]. Several
ndings suggest that GnRH-I plays regulatory roles
in processes prerequisite for decidualization and
Fig. 3. Topography of amino acid sequences of the three pig GnRH receptors type-I GnRHR (A), type-II GnRHR-5TM (B) and type-II GnRHR-
7TM (C). Adapted to Weesner and Matteri [106] and Neill et al. [130].
trophoblast invasion in humans [115]. Moreover, GnRH-
I plays various neuromodulatory roles: it is present in the
nervus terminalis, a neural plexus in the chemosensory
mucosa of the nasal cavity, where it has obviously the
function to modify olfactory information, perhaps at
reproductively auspicious times [28]. The physiological
roles of GnRH-I forms remain poorly understood until
now[88]. Most information has been gained, however, on
GnRH-II, where the specic conformation stabilizes the
peptide compared to GnRH-I. The conservation of the
GnRH-II structure >500 million years, unique location,
and differential expression levels of GnRH-II the type-II
receptor within and outside the brain in a distinct species
suggest that it has a variety of reproductive and non-
reproductive functions [8,10,21,43,88] (Table 2).
As mentioned above, GnRH-II has a lowafnity to the
type-I receptor. Therefore, it seems to be reasonable to
reassess whether functions that have been ascribed to
GnRH-I are might be caused by GnRH-II [130]. The
neuromodulatory roles outlined for GnRH-I were also
found for GnRH-II [18,125]. The conservation of the
GnRH-II structure may indicate more a function as
neurotransmitter which is involved in sexual behavior,
than that of a hormone which induces gonadotropin
release [21]. However, the only established function of
GnRH-II is the inhibition of M currents (K
+
channels) in
amphibian sympathetic ganglia mediated by binding to a
receptor that is a homologue to the primate type-II
GnRHR [21,134]. This function may support a role of
GnRH-II as neuromodulator that seems to be a common
and old function of GnRHvariants. It has been shown, for
example, that these peptides are able to stimulate
simultaneous spawning of individuals in a population,
and in this way assure more successful fertilization in
species that release their gametes into the water in which
they live [15]. In vertebrates, however, there is a high co-
localization of type-I receptors and type-II receptors in
pituitary gonadotropes. Therefore, GnRH-I and GnRH-II
may operate in concert to control the release of FSH and
LH. Silencing of the type-II GnRHR can be, however,
accommodated by GnRH-II signalling through the type-I
receptor with a distinctly different prole [11].
Is one of the GnRHs able, however, to act as an
specic FSH-releasing hormone (FSH-RH)? This issue
became the subject of speculation from the beginning of
the experimental GnRH story [83,135,136]. An FSH-
RH must be capable for selectively stimulating FSH
over LH or at least be a more potent releaser for FSH
than of LH which should result in a discordant FSH
secretion. GnRH-II has been designated as FSH-RH
[84,85]. On the other hand there is no study conrming
that the peptide is present in the pituitary portal blood
[137]. The presence, however, must be seen as a
prerequisite to exert a gonadotropin-releasing function.
Other studies using rat hemi-pituitary cultures showed
that only lGnRH-III stimulates FSH release at much
lower doses than those required for LH [86,138]. More
recent results conrmed the conclusion that lGnRH-III
or a closely related peptide was really the wanted FSH-
RH [46,87,139]. However, there is no consensus if
lGnRH-III functions as FSH-RH or not in mammals
[11] because others [29,140,141] were not able either to
isolate lGnRH-III from various mammalian brains or to
stimulate specically FSH secretion by this variant.
Recently, it was summarized that lGnRH-III has no
endocrine activity in mammals [142]. Therefore, it
seems to be necessary to proof the question again in
animal models, e.g. in sheep, in which the pituitary is
disconnected from the brain. This model would
eliminate problems in differentiating effects caused
by other peptides [21]. Apart from the gonadotropin-
releasing effects by GnRH-I to GnRH-III, and [Hyp
9
]-
GnRH, it was reported that wfGnRH(Table 1) increased
gonadotropin/TSHalpha subunit RNA expression in
dispersed rainbow trout pituitary cells [38] whereas
most of GnRH variants showed a very low activity in
stimulating gonadotropin release or ovulation [3].
For GnRH-II a role as an coordinator between food
intake and energetic condition, respectively, and repro-
ductive behavior was hypothesized. This function of
GnRH-II may result in mating under optimized condi-
tions [31,143]. It would be interesting to examine the
level of type-II GnRHRs expressed in that species and to
test these effects of GnRH-II in other mammals. Both
GnRH systems, the GnRH-I/type-IGnRHR and the
GnRH-II/type-II GnRHRsystem, likelyevolved together
and act in a synergistic manner. Because nutrition and
Table 2
Tissue distribution and proposed physiological functions for GnRH-II
Tissue Proposed function
Central and peripheral
nervous system
Neuromodulation
Mediobasal hypothalamus Control of gonadotropin secretion
Limbic structures Stimulation of reproductive behavior
Placenta Regulation of endocrine functions
Endometrium
Breast
Ovary
Tumors of the female
reproductive tract
Inhibition of cell proliferation
Normal T cells,
tumor T cells
Stimulation of cell
adhesion and migration
Adapted to Limonta et al. [8].
reproduction are closely linked, it may be plausible that
during evolution rst the more ancient GnRH form, i.e.
GnRH-II, served all functions that coordinated energy
and reproduction. Later GnRH-I usurped many of the
non-behavioral functions as the interactions between the
pituitary and gonads in mammals.
It may be a disadvantage that most of the studies on
the HPG axis were conducted in domesticated, well-fed
laboratory mammals which may or may not even
produce GnRH-II. Furthermore, it should be not
surprising that results of examinations of natural GnRH
variants in vivo and in vitro may differ from one species
or cell type to another. While it is becoming evident that
some species do not have a conventional type-II
GnRHR system, specic GnRH-II responses have been
observed in certain cell types. To solve this problem, the
demonstration of a functional GnRH-II/type-II receptor
system in cells lacking the type-I-receptor is needed
[115]. Recently, such system was explored that exists in
human endometrial, ovarian and breast cancer cells
probably enabling the therapeutic use of GnRH-II to
reduce proliferation of those not mediated by the type-I
receptor [126].
6. Use of GnRH-I and its natural forms in farm
animals
There is a growing interest to increase the efciency
of reproductive performances of farm animals, towards
of the inherited genetic potential [144]. The develop-
ment of systems to control ovarian function and
reproductive efciency with GnRHs and other phar-
maceutical agents has to be founded on an under-
standing of induced physiological effects and ability to
capture any advantage by good management of the farm
unit [145]. The pharmacological basis for the ther-
apeutic use of GnRH derives from its physiological
effect of stimulating gonadotropin release [146]. The
use of GnRH-I and synthetic analogues is common in
domestic animal production systems. The evaluation of
agonist potency depends largely on the model used and
wide varying potencies are reported for the same
agonist. The design of analogues has focused on
improving the receptor-binding and subsequent activa-
tion for agonists as well as on their increased resistance
to degradation by peptidases. In veterinary medicine the
most widely used agonists are the natural decapeptide,
buserelin and deslorelin [146]. Likewise, other syn-
thetic analogues have reached practical importance
(Table 3).
The single treatment by a small dose of the GnRH-I
agonist or its sustained release in low quantities will
induce, after binding to the receptor and internalization
of the ligand/receptor complex, a transient insensitivity
to stimulation in gonadotrophs. The type-I GnRHR is
replenished within several hours and this restores the
responsiveness of cells to GnRH. Unlike that antago-
nists may not provoke the initial stimulatory effect of
Table 3
Amino acid sequences of selected GnRH-I agonists and antagonists used/recommended for use in humans and farm animals
Amino acid
1 2 3 4 5 6 7 8 9 10
GnRH-I pGlu His Trp Ser Tyr Gly Leu Arg Pro Gly.NH2
Agonists
Lupron pGlu His Trp Ser Tyr D Leu Leu Arg Pro NEt
Zoladex pGlu His Trp Ser Tyr D Ser (tBu) Leu Arg Pro Gly.NH2
Supprelin pGlu His Trp Ser Tyr D His (ImBzl) Leu Arg Pro Gly.NH2
Synarel pGlu His Trp Ser Tyr D Nal Leu Arg Pro Gly.NH2
Triptorelin pGlu His Trp Ser Tyr D Trp Leu Arg Pro Gly.NH2
Buserelin pGlu His Trp Ser Tyr D Ser (tBu) Leu Arg Pro NEt
Goserelin pGlu His Trp Ser Tyr D Ser (tBu) Leu Arg Pro AzGly
Deslorelin pGlu His Trp Ser Tyr D Trp Leu Arg Pro NEt
Antagonists
Cetrorelix D Nal D Cpa D Pal Ser Tyr D Cit Leu Arg Pro D Ala. NH2
Ganirelix D Nal D Cpa D Pal Ser Tyr D hArg (Et)2 Leu D hArg (Et)2 Pro D Ala. NH2
Abarelix D Nal D Cpa D Pal Ser N Me Tyr D Asn Leu Lys (iPr) Pro D Ala. NH2
Antide D Nal D Cpa D Pal Ser Lys (Nic) D Cit Leu Lys (iPr) Pro D Ala. NH2
Teverelix D Nal D Cpa D Pal Ser Tyr D hCit Leu Lys (iPr) Pro D Ala. NH2
FE 200486 D Nal D Cpa D Pal Ser Aph (Hor) D Aph (Cba) Leu Lys (iPr) Pro D Ala. NH2
Nal-Glu D Nal D Cpa D Pal Ser Arg D Glu (AA) Leu Arg Pro D Ala. NH2
Bold letters: primary structure of GnRH-I and identical amino acids in analogues. Adapted to Millar et al. [17].
agonists and their blocking effects are immediate but
just as reversible [147].
Previously, one of the main objectives to use GnRH-I
and its synthetic agonists was to induce in females
reduced fertility or even infertility using the LH-
releasing and ovulation-inducing properties. The
development of pro-fertility treatments resulted in
several therapies, e.g. induction of ovulation, treatment
of cystic ovarian disease and prevention of embryonic
mortality. Numerous studies have been carried out with
GnRH-I and agonists in different climates, production
systems, reproduction status, etc. and sometimes
combined with other hormonal treatments. In this
context it is sometimes difcult to draw general
conclusions on treatment efcacy in several species
[146]. For several reasons the situation in the GnRH-
treated cattle needs special interest but conclusions are
to drawn parallel for sheep, horses and pigs.
GnRH-I agonists originally developed to treat
infertility show paradoxically inhibited reproduction
when given in high doses or even continuously. This
effect is exploited in anti-fertility methods of treatment.
There are, however, different ways that GnRH-I
analogues can be employed to inhibit reproduction
by direct suppression of the HPG axis at the level of the
gonadotroph: (1) chronic administration of agonists
causing down-regulation of type-I GnRHR and desen-
sitization of pituitary gonadotrophs; (2) immunization
against GnRH-I resulting in the neutralization of
GnRH-I in the hypophyseal portal blood by antibodies
and (3) the use of antagonists to block receptors to
occupancy by endogenous GnRH-I [147].
Among the pro-fertility treatments the therapy of
bovine cystic ovarian disease by GnRH was undertaken
in numerous studies [145,148150]. Although com-
monly used in veterinary practice the fertility of treated
cattle remains very poor after stimulating ovulation/
luteinisation of a new follicle and luteinisation of the
cyst. A better understanding of the syndrome is
probably required to improve fertility and not only to
restore normal fertility [146]. Another complex of
treatments by GnRH-I is directed to the support of
ovulation process. Data from treatments of various
species underline the importance of additional GnRH
on the day of insemination to improve the chances of
successful pregnancy. It is the common aim of the
treatment to stimulate luteinisation and early production
of luteal progesterone [151155]. When comparing the
effects of different studies it was summarized [146] that
the lower the background fertility in the herd, the higher
the percentage improvement. Given a satisfying fertility
in the herd the results may be equivocally.
In cattle but also in other species numerous
experiments were performed to enhance the luteal
function and improve the pregnancy rate by a GnRH
treatment approximately during the periods of maternal
recognition of pregnancy [156161]. Recently, it was
summarized that the balance of different data suggests
that post-insemination GnRH treatments are benecial
but exact validations of physiological and environ-
mental variables have to be executed in each treated
species [146]. A further aim of GnRH treatment is
directed to override the delayed resumption of ovarian
cyclicity in cows post-partum. It was stated [162] that
research on this topic seems to have lapsed in the last
decade when real time ultrasound was available for on-
farm diagnosis of ovarian status.
The treatment of seasonally anestrous animals, e.g.
sheep, by GnRH has reached only a limited importance
although scientically solved by applying multiple low-
dose injections of an agonist [163,164]. Reasons were
seen in the necessity to prime the animals by
progesterone to simulate a luteal-like period of cycle
followed by the peptide treatment which will increase
unfavourably the costs of treatment.
Use of GnRH as an integral part of synchronising
regimens where is given 7 days before PG an then again
4860 h after PG appears to be effective in increasing
the synchrony of ovulation in controlled breeding
programmes [165170]. Whereas the rst injection is
important for the prolongation of luteal phases in those
animals treated late in the cycle, the main synchronising
effect seems result from the second GnRH injection.
This injection advanced the timing of ovulation and the
subsequent rise in progesterone concentrations by an
average of 24 h relative to cows just receiving a GnRH-
PG regimen [146].
The other complex of methods that use GnRH-I
analogues is summarized as anti-fertility technologies.
Long-term fertility control agents are of interest for the
management of reproduction in humans, domestic
animals and wildlife. This topic is important in the
management of farm animals but from both the
behavioral and fertility standpoint, as is the case with
domestic dogs and cats [147]. A rst group of methods
is using the paradox effect of continuous GnRH on
reproductive function. The continuous availability of
the peptide is reached mainly by depot formulations.
The desensitization of the gonadotrophes is followed
by a decline in ovarian and testicular functions,
respectively. This phenomenon is extensively applied
in clinical medicine for the treatment of a wide range
of disease [12]. Depot GnRH-I agonists are now
increasingly used in animal treatments [171174].
Recently [147] it was reviewed that agonists when
released over periods of some months showed a high
contraceptive rate both in veterinary regimes to treat
farm and domestic animals. The fertility control by
GnRH-I agonists has some advantages when compared
with traditional surgical sterilization methods. One
advantage is that contraception is affected after a single
injection without the need for use of adjuvants.
Numerous studies demonstrated a positive relationship
between the dose of agonist and the duration of
suppression but the reasons are still unknown. Recent
results indicated that a continuous inuence of a depot
GnRH-I agonist over a period of more than 1 week will
impair the development of bovine follicles and oocytes
[175].
Moreover, a wide range of variation was observed
between individual animals in the interval to resumption
of oestrous cycles after treatment and in the function of
testes, respectively. A recent review [176] discussed the
effects observed in growing and mature boars of a long-
acting agonist treatment. Results of various studies
[177,178] suggested that the treatment may be an
effective means of reducing the taint in boar meat. In
contrast to the response detected in most other species
results were described that a GnRH-I agonist treatment
induced an increase in the reproductive function. The
treatment may be, for example, effective to accelerate
puberty in calves [176,179].
Other possibilities to control fertility by GnRHare the
treatments by antagonists and the immunization against
the peptide resulting in its neutralisation in the portal
blood by antibodies. Both methods are in the phase of
development but they may have potential. The immu-
noneutralisation of endogenous GnRH-I may prove to be
an useful management tool in keeping both female and
male animals [176]. The method has the advantage that it
does not have a stimulatory acute phase [180185]. One
disadvantage of vaccination is, however, the inconsistent
and variable immune response of treated animals. Novel
types of anti-GnRHvaccines are nowunder development
to reduce the variation.
GnRH-I antagonists (Table 3) also inhibit the
reproductive system through competition with endo-
genous peptide but the doses required to block
gonadotropin release are higher than that of the
comparable agonist treatment [186191]. Efcient
delivering systems or the development of non-peptide
orally active antagonists are likely to replace agonists as
they avoid the undesirable stimulation that precedes
desensitization [12]. Until acceptable technologies are
available, the use of GnRH-I antagonists is especially to
recommend for short-term treatments, e.g. down-
regulation of the pituitary during IVF treatment regimes
[147].
In brief, GnRH-based methods to manage fertility in
farm animals have potential but the widespread
application of these technologies is limited to date.
Compared with the possibilities to apply GnRH-I and its
synthetic analogues knowledge is only scarce on
possibilities for the natural GnRH-I analogues. The
main reason is that the physiological roles of GnRH-II
and its receptor, and the roles of other pairs, relative to
GnRH-I and its receptor are unknown [30]. Also, other
facts may contribute, like the poor uniformity of
occurrence [192] and reception [33] of GnRHs in
mammals which make the development of suitable
models more difcult. It seems to be nearly impossible
to evaluate in vivo-effects of GnRHs beside that of
GnRH-I. From data available at present, the following
hypotheses should be executed in various models:
Natural GnRHs unlike GnRH-I present in farm
animals are residues of a long developmental process.
They have no or less independent importance for
basic functions of mammalian reproduction. GnRHs
may intensify GnRH-I-mediated effects within the
HPG axis. The contributions of GnRHs are still
unknown but may vary between species, periods of
the reproductive cycle, tissues and environmental
factors, etc.
GnRHs like GnRH-II, lGnRH-III or GnRH-III have
functions within the neuroendocrine regulation of
reproduction distinct from GnRH-I functions as
discussed for the specic release of FSH [84
87,139,193]. This is supported by the fact that
chronic administration of a GnRH-I shows a
differential response of pituitary gland and gonads
to the medication [194], even in ruminants [156].
GnRHs unlike GnRH-I show specic effects which
result from the past like the combination of external
factors from the environment with neuroendocrine
mechanisms of regulation of reproductive function. In
mammals, where, for example, GnRH-II is expressed
at signicantly higher levels outside the brain, the
peptide may participate, more than GnRH-I [10] in
the induction of mating behavior. Ovarian steroids
and their antagonists were reported to affect
differentially the expression of GnRH-I and GnRH-
II in human granulosa cells at the transcriptional level
[195]. Both GnRH-II [9] and lGnRH-III [142] showed
a direct antiproliferative effect on various cancer cell
lines. Superactive analogues of that may, therefore,
useful in specic therapeutic regimes in humans,
instead of long-term administered GnRH-I agonists
[9]. An inclusion of GnRHs in treatment schedules of
farm animals seems to be possible.
To execute the preceding hypotheses, some general
premises should be realized. The rst step is to complete
the listing of presence of various GnRH forms in one
species, in one tissue or expressed in one cell type. Next,
the spectrum of analytical methods to characterize
highly specically the biosynthesis, release of single
variants in vivo and in vitro, and reception has to be
replenished. Thus, animal models have to be created
which may demonstrate specic effects of GnRH
variants independent on the other forms, in particular
GnRH-I, which may effect negligible low [21].
However, the function of GnRH-II as coordinator
between nutrition and reproduction [143] should be
explored in experimental studies on animals fed at
different levels. Generally, development and use of
potent synthetic analogues (agonists and antagonists) of
promising GnRHs-like [D-Ala
6
]-GnRH-II and Trptor-
elix-1 [20] and [D-Arg
6
]-GnRH-II-aza-Gly(10)-NH2
[196] may contribute to the exploration of functions of
non-mammalian GnRHs in mammals. On the basis of
these diverse studies a further progress in the
development of effective and animal friendly applica-
tions for natural GnRHs might be expected.
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