# Gonadotropin releasing hormones and their receptors in avian species

Grégory Bédécarrats, Mamiko Shimizu, Daniel D. Guemene

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Grégory Bédécarrats, Mamiko Shimizu, Daniel D. Guemene. Gonadotropin releasing hormones and their receptors in avian species. The Journal of Poultry Science, 2006, 43, pp.199-214. 10.2141/jpsa.43.199. hal-02655007

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Gonadotropin Releasing Hormones and their Receptors in
Avian Species

Grégoy Y. Bédécarrats<fnref n="1" />, Mamiko Shimizu<fnref n="1" /> and Daniel Guémené<fnref n="2" />

In all vertebrates, gonadotropin releasing hormone (GnRH) is a key component of the hypothalamo-pituitary-gonadal axis. In birds, two distinct forms of GnRH (cGnRH-I and cGnRH-II) have been characterized, and a third immunoreactive form similar to lamprey GnRH-III has been reported. Each GnRH is produced by a different population of neurons. cGnRH-I is predominantly synthesized in cells located in the preoptic and septal areas, whereas cGnRH-II and -III are produced in neurons located mainly in the mesencephalon and around the third ventricle, respectively. Although GnRH neurons have been shown to project fibers to the median eminence, it is still unclear whether all three peptides are released in the hypothalamo-pituitary portal vascular system. Nonetheless, *in vitro* as well as *in vivo* experiments confirmed that they all have the ability to induce the release of gonadotropins. In the anterior pituitary gland, GnRHs mediate their effects by binding to specific G-coupled protein receptors, and in chickens, two different receptors have been characterized (cGnRHR-I and cGnRHR-II). In addition to the full-length receptors, several splice variants have also been detected. Although the physiological relevance of these variants is not known, they may participate in the regulation of intracellular signaling. While the expression of the full-length cGnRHR-I appears to fluctuate during an ovulatory cycle, levels remain constant between reproductive stages in chickens. In contrast, pituitary levels of cGnRHR-II mRNA are directly correlated with the reproductive status of the bird. Whether these two GnRHRs are expressed by the same cells in the anterior pituitary gland is not known. However, since luteinizing hormone (LH) and follicle stimulating hormone (FSH) are produced by two different cell populations in chickens, it is possible that different combinations of GnRHs and GnRHRs differentially mediate the synthesis and release of these gonadotropins.

**Key words** : GnRH, GnRH receptor, reproduction

J. Poult. Sci., 43 : 199-214, 2006

Introduction

As for most vertebrates, reproduction in avian
species is controlled by the hypothalamo-pituitary-
gonadal axis. The physical and physiological events
associated with the reproductive cycle of birds have
been reviewed and described in detail elsewhere
(Etches, 1996; Chaiseha and El Halawani, 2005).
Briefly, upon photostimulation, gonadotropin releas-

ing hormone (GnRH) is released by the hypothala-
mus and stimulates the synthesis and release of
gonadotropins (luteinizing hormone LH and follicle
stimulating hormone FSH) by the anterior pituitary
gonadotropes. In turn, LH and FSH stimulate the
development of the gonads, and activation of this
axis ultimately results in the initiation and mainte-
nance of egg/sperm production. In addition to
GnRH, the recent discovery of a gonadotropin in-

<footnote n="1">¹ Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, N1G2W1, Canada</footnote>

<footnote n="2">² Unité de Recherches Avicoles, INRA, 37380 Nouzilly, France</footnote>
hibiting hormone (GnIH) has allowed the elabora-
tion of new theories on the hypothalamic control of
gonadotropins (Tsutsui *et al.*, 2000). The implica-
tion of GnIH in birds' reproduction is the subject of
a separate review published in this issue of the
Journal of Poultry Science.

In both vertebrates and invertebrates, multiple
forms of GnRH have been isolated, and with the
exception of the Octopus vulgaris (Iwakoshi et al.,
2002), they all correspond to decapeptides with a
relatively well conserved sequence (Somoza et al.,
2002). Based on brain localization as well as species
distribution, GnRH peptides can be classified into
three major categories ; a hypothalamic specific
form (GnRH-I) present mainly in the preoptic area,
an “ancient” form conserved throughout species
(GnRH-II) present mainly in the mesencephalon,
and a GnRH-III form present in the telencephalon
of some species (Somoza et al., 2002 ; Millar et al.,
2004). As for GnRH, multiple forms of its receptor
(GnRHR) exist, and although most types of GnRH
are able to bind and activate every GnRHR, differ-
ences in affinity and specificity are observed (Barran
et al., 2005). Among vertebrates, GnRHRs can be
subdivided into mammalian and non-mammalian
receptors. The most striking difference between the
two being the lack of an intracellular C-terminal tail
in the mammalian forms. This is of importance
since the C-terminal tail was shown to be responsible
for faster internalization (Pawson et al., 1998). In
addition, several specific micro-domains have also
been shown to harbor ligand specificity (Wang et al.,
2004).

Since reproductive efficiency is central to a suc-
cessful poultry industry, GnRH and its effects on the
reproductive axis have been extensively studied in
avian species and were previously reviewed by Dunn
and Millam (1998) and Sharp et al. (1998). How-
ever, compared to mammals, few studies have
focused on the structure/function of the GnRHRs
in birds. This review summarizes earlier work on
the identification and distribution of avian GnRHs,
the effects of GnRHs on gonadotropins, and the
characterization and function of GnRHRs.

Identification of GnRH Variants
in Avian Species

In birds, three major types of GnRH have been
isolated. However, whether all three forms directly

regulate pituitary gonadotropes remains controver-
sial.

Evidence supporting the existence of GnRH in
chickens was originally obtained from immunologi-
cal studies (King and Millar, 1979), and cGnRH-I
was first purified from chickens' hypothalami
(Miyamoto et al., 1982). Its structure and amino
acid sequence were further determined (King and
Millar, 1982; Miyamoto et al., 1983), and it was
shown to differ from the mammalian form by a
substitution of an Arg with a Gln at position 8. As
its mammalian counterpart, cGnRH-I is encoded by
a gene composed of four 4 exons, leading to the
synthesis of an 89 amino acid prepropeptide (Dunn
et al., 1993). In addition, a splice variant with
retention of intron I which could result in a normal-
ly translated product was also identified (Sun et al.,
2001a). Processing of cGnRH-I prepropeptide and
the possible role of its cryptic associated peptide
have been previously reviewed (Dunn and Millam,
1998), including the specific enzymatic cleavage of
the signal peptide, and the pyrolisation and amida-
tion of the N- and C-terminal residues, respectively.
The function of the associated cryptic peptide still
remains to be determined, and although it may allow
for normal processing of cGnRH-I, an endocrine
role cannot be excluded.

In 1984, Miyamoto *et al.* identified a second GnRH (cGnRH-II) from chicken hypothalamic extracts. This cGnRH-II differs from cGnRH-I at position 5, 7 and 8. Interestingly, this form appears to be fairly conserved among vertebrates, and has been detected in several extra-hypothalamic tissues. Thus, it is thought to be the most ancient form of GnRH with multiple possible functions (Millar *et al.*, 2004). However, although GnRH-II sequence is ubiquitous, cloning of the chicken gene revealed that the prepropeptide differs greatly between mammals and avian species (Ikemoto and Park, 2005). In addition, as the cGnRH-II gene partly overlaps with another gene (MRPS26), five different transcripts have been identified, with three of them possibly generating a mature decapeptide (Ikemoto and Park, 2005). Based on immunocytochemistry studies, a third form of GnRH (similar to lamprey GnRH-III) has also been detected in the brain of songbirds (Bentley *et al.*, 2004). Although the biochemical structure of this new form of GnRH has not been characterized, it appears to bind specifi-
cally to antibodies raised against lamprey GnRH-III. Furthermore, preabsorption of the lamprey GnRH-III antiserum with cGnRH-I and II did not reduce binding to immunoreactive cells (Bentley *et al.*, 2004).

Distribution of GnRH Variants

Brain distribution, presence in the median eminence, and release in the hypothalamo-pituitary portal vascular system are all critical factors that dictate physiological relevance of GnRHs. Unfortunately, most localization studies rely on the use of antibodies with various degrees of specificity and sensitivity, and the distribution of cGnRHs still remains controversial.

The ontogeny of GnRH-I neurons in birds have previously been extensively reviewed by Norgren (1996) and Dunn and Millam (1998). In the chicken, as reported in many other species, GnRH-I neurons originate from the olfactory placode where they can be detected as early as embryonic day 3 to 5 (Murakami *et al.*, 1991; Norgren *et al.*, 1992; Sullivan and Silverman, 1993). These early GnRH-I cells then migrate into the brain area along the olfactory nerve, and reach the septum by embryonic day 7. From then on, cell bodies reach their adult location in the brain, and start projecting their axons toward the median eminence. Interestingly, the concentration of cGnRH-I in the chick brain decreases between embryonic day 8 and 12, and then starts increasing after day 17 (Millam *et al.*, 1993a). It is possible that this reduction in immunoreactive GnRH-I results from gonadal steroid negative feedback during the maturation of the hypothalamo-pituitary-gonadal axis. In contrast to cGnRH-I, cGnRH-II levels remain extremely low in the brain of chicks until embryonic day 14, drastically increasing up until hatch (Millam *et al.*, 1993a). To date, no evidence suggests that cGnRH-II neurons also originate in the olfactory placode. However, cGnRH-II antisera used in immunohistochemical studies may lack the sensitivity to detect extremely low amounts of this peptide. Sensitivity of antiserum is a critical issue and is most likely responsible for the lack of data regarding the presence of cGnRH before the formation of the olfactory placode. However, using more sensitive techniques such as RT-PCR and *in situ* hybridization, Witkin *et al.* (2003) detected the expression of cGnRH-I gene

in chicken embryos along the primitive streak before gastrulation, followed by the anterior neural ridge and the olfactory placode. These observation strongly suggest that cGnRH-I precursors may be committed as early as HH stage 4.

In adults, although early immunohistological studies using antibodies raised against mGnRH confirm the presence of cGnRH perikarya in the hypothalamus and other brain areas, as well as the presence of cGnRH fibers in the median eminence of several avian species, specific distribution of cGnRH-I and cGnRH-II neurons has not been determined (Dunn and Millam, 1998). Following the elucidation of the biochemical structure of both cGnRHs, specific antibodies against cGnRH-I and cGnRH-II were made available, and in 1988 Mikami *et al.* were the first to report a different distribution of immuno-reactive cGnRH-I and II cells in the quail and chicken brain. The two peptides appear to be present in two different populations of neurons with cGnRH-I perikarya located mainly in the preoptic and septal areas of both quail and chickens (Mikami *et al.*, 1988; Van Gils *et al.*, 1993), and the periventricular nucleus of chickens (Mikami *et al.*, 1988). In contrast, cGnRH-II perikarya were observed mainly in the rostral end of the mesencephalon around the occulomotor complex (Mikami *et al.*, 1988; Van Gils *et al.*, 1993). Similar results were also reported in the turkey (Millam *et al.*, 1993b), where cGnRH-II neurons are located around the occulomotor complex extending to the mesencephalon. Kuenzel and Blahser (1991) further established the specific location of six major groups of cGnRH neurons throughout the brain. These include the olfactory bulb, olfactory tubercle, nucleus accumbens, three nuclei in the septal/preoptic hypothalamus (lateral septal and lateral ventricle areas, pallial commisure, preoptic/paraventricular nucleus), lateral anterior thalamic nucleus, and occulomotor complex (midbrain). However, these authors used an antibody raised against mammalian GnRH, and were thus not able to discriminate between cGnRH-I and cGnRH-II neurons.

To be physiologically relevant at the level of the pituitary, a given cGnRH needs to be present in the median eminence of the hypothalamus. Immunohistochemical evidence clearly demonstrates that cGnRH-I fibers project to the median eminence, especially cells from the pallial commisure. On the
other hand, whether axons from cGnRH-II neurons
reach the median eminence is controversial, and
although Mikami *et al.* (1988) and Millam *et al.*
(1993 a) failed to detect any cGnRH-II im-
munoreactive fibers in the median eminence of quail
and chickens, Van Gils *et al.* (1993) and D’Hondt *et
al.* (2001) were able to. Radioimmunoassay meas-
urement of cGnRHs in chicken brain confirmed
immunohistochemical evidence that cGnRH-I is
significantly more abundant than cGnRH-II in the
hypothalamus (Katz *et al.*, 1990; Sharp *et al.*,
1990), and that cGnRH-II is not (Sharp *et al.*,
1990) or barely (Katz *et al.*, 1990) present in the
median eminence. Similarly, in Japanese quail,
cGnRH-I concentrations in individual median emi-
nences are far greater than that of cGnRH-II
(Millam *et al.*, 1998). However, using the same
antibody as Van Gils *et al.* (1993) and D’Hondt *et
al.* (2001), Clerens *et al.* (2003) detected and
purified cGnRH-II from the median eminence of
quail. In addition to cGnRH-I and II, a new im-
munoreactive cGnRH-III similar to lamprey
GnRH-III has also been detected in the brain of
songbirds (Bentley *et al.*, 2004), and cGnRH-III
neurons appear to be present in multiple areas with
a higher density around the third ventricle. In-
terestingly, axons were also shown to project to the
median eminence. It is worth mentioning that lam-
prey GnRH-III immunoreactive neurons were dis-
tinct from cGnRH-I and II cells (Bentley *et al.*,
2004).

In order to exert their effects on gonadotropins,
cGnRHs need to reach the pituitary gland, and thus
be released in the hypothalamo-pituitary portal
blood system. Perifusion of hypothalamic slices
containing the median eminence were shown to re-
lease GnRH in quail (Millam et al., 1984), and both
cGnRH-I and II were shown to be present in the
posterior pituitary of turkeys (Rozenboim et al.,
1993). However, as reported by Millam et al.
(1998), the release of cGnRHs from hypothalamic
explants in perifusion may not necessarily represent
the median eminence release, but may rather repre-
sent leakage from other hypothalamic structures.
Interestingly, as reported in several mammalian spe-
cies, mRNAs for cGnRH-I (Sun et al., 2001) and
cGnRH-II (Ikemoto and Park, 2005) were also
detected in the pituitary gland of chickens. Al-
though these levels appear to be minimal, this raises

the possibility that GnRHs may act as paracrine/
autocrine factors within the pituitary.

Control of GnRHs Synthesis and Release

GnRHs are mainly associated with the develop-
ment and function of the reproductive axis in avian
species. Thus, the control of their synthesis and
release will be discussed in the context of a repro-
ductive cycle. Although birds can sexually mature
spontaneously, the hypothalamo-pituitary-gonadal
axis can be prematurely stimulated by increasing the
photoperiod (photostimulation), with a transfer
from short (7 h) to long (14 h) photoperiod provid-
ing maximum stimulation in chickens (Lewis et al.,
2002). During the onset of puberty or acquisition of
sexual maturity, the abundance of cGnRH-I mRNA
and peptide increase in the hypothalamus of chick-
ens (Sharp et al., 1990; Dunn and Sharp, 1999) and
the basal hypothalamus of starlings (Dawson et al.,
2002). An increase in GnRH release can also be
observed in superfused hypothalamic slices from
photostimulated quail (Millam et al., 1984), and this
increase occurs within 23 h post photo-stimulation in
hypothalamic explants of quail (Perera and Follett,
1992). At a histological level, the number of
cGnRH-I fibers were shown to increase in the hypo-
thalamus of breeding house sparrows when com-
pared to non-breeding and juvenile birds (Stevenson
and Macdougall-Shackleton, 2005). Similarly, in
the same study, cGnRH-II fibers were also more
abundant in the pre-optic area of breeding house
sparrows, suggesting a plastic reorganization of
neurons during sexual maturation. However, a role
for cGnRH-II during sexual maturation still
remains controversial, and Sharp et al. (1990) did
not observe any changes in the hypothalamic con-
tent of peptide during the onset of puberty in cock-
erels. Nonetheless, despite the controversy sur-
rounding cGnRH-II, it is clear that exposure to light
controls the release of GnRH via hypothalamic
photoreceptors (Sharp, 2005). Unfortunately, these
receptors have been elusive and still remain to be
identified. As suggested by Wilson (1997), thyroid
hormones may also play an important role in the
control of photoperiodism and the associated
changes in GnRH-I synthesis and release during a
reproductive cycle in sparrows. This was confirmed
by the fact that thyroidectomy abolishes the increase
in GnRH-I observed after photostimulation (Daw-
son *et al.*, 2002). Interestingly, the mechanisms by which thyroid hormones control GnRH neurons seem to reside within the hypothalamus, and photostimulation stimulates the conversion of T4 to T3 in the mediobasal area in quail (Yoshimura *et al.*, 2003). In turn, this increase in T3 induces mor- phological changes in the median eminence, allow- ing GnRH neurons to contact the peri-capillary space (Yoshimura, 2004). These findings have allowed the elaboration of new theories on how light stimulates the release of GnRH, thus stimulating the hypothalamo-pituitary-gonadal axis (for review : Yoshimura, 2004 ; Sharp, 2005). In adult hens, sexual maturity is characterized by the beginning of egg-laying. Ovulation in avian species is induced by an LH surge caused by positive feed-back from progesterone released by the dominant follicle (Wilson and Sharp, 1976). Whether GnRH plays an important role in mediating the progesterone effect on LH surges remains uncertain. In chickens and turkeys, laying rate progressively decreases with age, and hens will either enter a photorefractory period or express incubation behavior. No decrease in hypothalamic cGnRH-I mRNA or peptide con- tent occur during the progressive cessation of egg- laying in chickens (Sharp *et al.*, 1992 ; Ciccone *et al.*, 2005). However, a significant decrease in mRNA levels was observed between laying and in- cubating hens (Dunn *et al.*, 1996), although this was not associated with changes in peptide content. In the turkey, a decrease in cGnRH-I and II peptide content in the hypothalamus was observed between laying or incubating hens and photorefractory hens (Rozenboim *et al.*, 1993). These observations are somewhat contradictory to the reports of Millam *et al.* (1989) and Millam *et al.* (1993 b), who showed that the hypothalamic content in cGnRH-I and II is the highest during incubation in the turkey. These discrepancies may be explained by the fact that different hypothalamic regions were investigated, and whether the median eminence was included. In addition, mRNA levels do not necessarily correlate with cellular peptide content and are often not rep- resentative of the amount of peptide released. In the quail, cGnRH-I concentrations decrease in the median eminence of aging birds, and although con- centrations in the preoptic area remain similar, cGnRH-I release is reduced in older birds (Ottinger *et al.*, 2004). Thus, it seems likely that different

mechanisms control gene expression, storage in the median eminence, and release of cGnRHs. A model for the effects of genotype dependent inputs from a biological clock on the secretion of GnRH in long and short day breeders has recently been proposed (Sharp, 2005). In this model, both stimu- latory and inhibitory inputs affect the release of GnRH, and the impact of these factors is under the control of an internal biological clock as well as under the influence of external stimuli such as pho- toperiod. However, in addition to the positive and negative inputs from the biological clock, ovarian steroids may play a major control role once active egg-laying is initiated (Figure 1).

Effects of GnRHs on Gonadotropins

Although it remains unclear whether all three GnRHs are released into the hypothalamo-pituitary portal blood system, several studies confirmed that they all have the ability to stimulate pituitary gonadotropes. In quail and chickens, both cGnRH- I and II stimulate the release of LH and FSH *in vitro* as well as *in vivo* (Hattori *et al.*, 1986), and in sparrows a homolog to lamprey GnRH-III can also stimulate the release of LH *in vivo* (Bentley *et al.*, 2004). However, their relative potency is debatable, and cGnRH-I was shown to be as potent as cGnRH- II in stimulating LH release *in vitro* from immature cockerel (Hattori *et al.*, 1986) and in out-of-lay turkey hens (Guémené and Williams, 1999) pituitaries. On the other hand, cGnRH-II appears to be the most potent LH releasing hormone on cultured dispersed chicken pituitary cells (Chou *et al.*, 1985; Millar *et al.*, 1986) and perifused pitu- itaries from males (Guémené and Williams, 1992 a) and incubating turkey hens (Guémeneé and Wil- liams, 1999). Similarly *in vivo*, cGnRH-II has been shown to be more potent than cGnRH-I in both chickens (Wilson *et al.*, 1989; Proudman *et al.*, 2005) and male turkeys (Guémené and Williams, 1992 a). However, no difference in potency was observed after injection of cGnRH-I and II in cock- erels (Chou *et al.*, 1985), while cGnRH-II was shown to be more potent in male turkeys (Guémené and Williams, 1992 a). On the other hand, cGnRH- I was as or more potent than cGnRH-II in the minutes following injection in turkey hens, while cGnRH-II was longer lasting (Guémené and Wil- liams, 1999). Although results from these studies
<figure><img src="image_3.png" /><figcaption>Fig. 1. A model for the interaction of cGnRHs with cGnRHRs, and the resulting effects on gonadotropin release. The relative input of each component is re-presented by the thickness of lines and arrows. Dotted lines correspond to putative inputs. A, corresponds to an immature reproductive state. At this stage, GnRH-I-II and -III synthesis and release by the hypothalamus is minimal, and all three peptides have potentially the ability to bind to GnRHR-I and -II to maintain low levels of gonadotropins. In turn, low levels of LH and FSH result in minimal hormone production from the gonads which maintain a positive/negative feed-back on the hypothalamus and the pituitary gland. B, during the acquisition of sexual maturity following photostimulation, levels of cGnRHs secretion by the hypothalamus increase, and activation of the cGnRHR-I results in an increase in LH release by the pituitary. Simultaneously, levels of cGnRHR-II increase in the pituitary followed by an increase in FSH release. Both gonadotropins then induce the maturation of the gonads which ultimately results in spermatogenesis and ovulation. Once mature, gonads secrete steroid hormones which further regulate the secretion of LH by both negative and positive feed-back, whereas gonadal hormones inhibit FSH release. C, as the active reproductive phase progresses, cGnRHs secretion by the hypothalamus decreases reducing the stimulation of LH through cGnRHR-I. In addition, levels of cGnRHR-II in the pituitary decrease resulting in a lack of FSH stimulation. At this stage, gonadal steroids' positive and negative feed-backs on LH release are sufficient to maintain successive ovulation and egg laying.</figcaption></figure>

appear to be contradictory, it is worth mentioning that species, sex, and reproductive status need to be taken into consideration. For example, intravenous injection of cGnRH-II is more effective in triggering the release of LH in hens than in cockerels (Sharp *et al.*, 1987), while the opposite is true in turkeys (Guémené and Williams, 1992 ab, 1999). Furthermore, perifused pituitaries from incubating turkey hens are more responsive to cGnRH-II than cGnRH-I whereas no difference in potency is observed with pituitaries from out-of-lay hens, and

pituitaries from laying hens failed to respond to cGnRH (Guémené and Williams, 1999). Interestingly in an earlier study, pituitaries from laying turkey hens were shown to respond to mammalian GnRH (Guémené and Williams, 1992 b).

Although cGnRHs are clearly able to stimulate the release of LH, their role in the control of FSH is more questionable. A positive effect of GnRH on FSH release was reported in dispersed chicken pituitary cells (Millar *et al.*, 1986) as well as *in vivo* and *in vitro* in quail (Hattori *et al.*, 1986). However,
little or no significant increase in circulating levels of FSH could be induced by injecting either cGnRH-I, -II or lamprey GnRH-III in chickens (Krishnan *et al.*, 1993; Dunn *et al.*, 2003; Proudman *et al.*, 2005). The discrepancy observed between these studies may result from the various degrees of specificity of radioimmunoassays used for the measurement of chicken FSH, as the assay used by Hattori *et al.* (1986) was shown to partly cross-react with LH (Sakai and Ishii, 1983). However, as mentioned for the control of LH secretion, sex, age and reproductive status may also be a determining factor. Most injections of GnRH that failed to induce the release of FSH were performed on non-photostimulated pullets (Dunn *et al.*, 2003), prepubertal pullets (Proudman *et al.*, 2005), and non-laying hens or immature cockerels (Krishnan *et al.*, 1993). Interestingly, an increase in circulating levels of FSH was observed when high doses of cGnRH-II were injected in 32 weeks old mature roosters (Proudman *et al.*, 2005). As described in the following chapter, large variations in GnRHR-II mRNA levels are observed in the pituitary gland of chickens during a reproductive cycle (Shimizu and Bédécarrats, 2005), and it is thus possible that varying amounts of GnRHRs differentially mediate the effect of cGnRHs on LH and FSH secretion. This is an attractive hypothesis (presented in Figure 1), as LH and FSH have been shown to be produced by two different populations of gonadotropes (Proudman *et al.*, 1999; Puebla-Osorio *et al.*, 2002). In ganders, intramuscular injection of Phe6-GnRH-Ethylamide (a GnRH agonist) between photostimulation and sexual maturity did not significantly affect circulating levels of LH (Sellier *et al.*, 1995). However, it did transiently decrease levels of testosterone, increase the fertility during the second half of a reproductive period, and partially prevented the natural decline in sperm production (Sellier *et al.*, 1995). Although FSH levels were not measured, it appears that the GnRH effects observed in the latter study were independent of the release of LH.

Nonetheless, the most convincing evidence regarding the relative physiological relevance of cGnRHs in chickens is probably the gonadal reduction observed after the active immunization of laying hens against cGnRH-I and not cGnRH-II, pointing at cGnRH-I being “the” gonadotropin releasing factor (Sharp *et al.*, 1990).

In birds as in mammals, GnRH secretion is pulsatile, and *in vitro* perfused hypothalamic sections release GnRH-I at a frequency of approximately 3 pulses per hour (Li *et al.*, 1994). Furthermore, the pulsatility center appears to be located in the medio-basal hypothalamus and the preoptic area (Li *et al.*, 1994). Pulsatility is also observed *in vivo* where GnRH pulse frequency can be deduced from profiles of circulating LH. In quail, LH pulses could be observed only after exposure to long days with a frequency of 1 pulse per 80 min (Urbanski, 1984). In turkey hens, pulsatile release of LH is observed before photostimulation with an average of 4 to 6 pulses per 23 hours. However, following photostimulation, basal levels significantly increase and pulses become less apparent (Chapman *et al.*, 1994). A lack of detectable discrete LH pulses in laying turkey hens has also been reported (Yang *et al.*, 1997). In broiler breeders, pulsatile LH release could be observed at an average frequency of 2 pulses per hour in males (Vizcarra *et al.*, 2004) and 1 pulse per hour in females at the end of a laying period (Senthilkumaran *et al.*, 2005). Interestingly, in male broiler breeders, the profile of FSH pulses appears to be independent from that of LH, again suggesting a different regulation between the two gonadotropins (Vizcarra *et al.*, 2004). Taken together, these studies suggest that, conversely to mammals, in which varying GnRH pulse frequency differentially regulates LH and FSH synthesis and release during an ovarian cycle (Levine and Ramirez, 1982; Haisenleder *et al.*, 1991; Bédécarrats and Kaiser, 2003), the initiation and maintenance of egg-laying in avian species is mainly associated with an increase in LH baseline concentrations rather than an increase in pulse frequency. Nonetheless, continuous GnRH exposure or stimulation at a frequency exceeding 1 pulse/30 min induces the desensitization of diced pituitary glands in perfusion (Guémené and Williams, 1992ab; Guémené and Williams, 1999), suggesting that as observed in mammals (Belchetz *et al.*, 1978), pulsatile GnRH is required to effectively stimulate pituitary gonadotropes.

## Isolation of GnRH Receptors and their Splice Variants in Avian Species

The fact that cGnRHs are able to specifically induce the release of LH and FSH *in vivo* as well as
in vitro, and that pituitary gonadotropes can be
desensitized by continuous stimulation suggests that
cGnRHs act through specific cell surface receptors.
Evidence of specific binding of cGnRH-I to chicken
pituitary cell membranes was first reported by Millar
and King (1983). Interestingly, the affinity of a rat
pituitary cell membrane preparation for cGnRH-I
appeared to be lower than for its mammalian coun-
terpart (Millar and King, 1983). Binding character-
istics of cGnRH-I to a chicken pituitary cell mem-
brane preparation were characterized by Kawa-
shima et al. (1992). The dissociation constant (Kd)
as well as the maximum binding (Bmax) appeared
to fluctuate with the reproductive stage as well as the
ovulatory status of the hen, suggesting changes in
the number of cell surface binding sites and the
existence of possibly more than one receptor. The
first cGnRHR (designated as cGnRHR-I in this
review) to be cloned was originally isolated from
adult cockerels (Sun et al., 2001 a). As in other
vertebrates, this receptor belongs to the G-protein-
coupled receptor (GPCR) family, with its charac-
teristic seven transmembrane domains. However,
the existence of at least two GnRHs within a species
suggests that cognate receptors are present. As a
matter of fact, many vertebrates from amphibians
and fish (Illing et al., 1999; Troskie et al., 2000;
Jodo et al., 2005) to mammals (Millar et al., 2001;
Neill et al., 2001; Morgan et al., 2003) possess more
than one receptor. Thus, it is likely that additional
GnRHRs also exist in avian species, an assumption
that has been supported by the assembled chicken
genome. We have recently cloned a novel cGnRHR
(designated as cGnRH-II, Figure 2, Genebank ac-
cession number : AY895154) from the pituitary
gland of a mature White Leghorn male (Shimizu
and Bédécarrats, 2005). Unlike mammalian Gn-
RHR genes which consist of 3 exons and 2 introns
(Zhou and Sealfon, 1994; Kakar, 1997), both
cGnRHR-I and II genes are composed of 4 exons
and 3 introns encoding a 375 and 419 amino acid
protein, respectively. At the amino acid level,
cGnRHR-II shares 53% homology with the cGn-
RHR-I and 51-65% homology with the amphibian
and fish receptors. As for other non-mammalian
receptors, cGnRHRs posses a carboxy-terminal tail
which has been shown to be responsible for de-
sensitization and rapid internalization (Pawson et
al., 1998; Blomenrohr et al., 1999; Pawson et al.,

2003).

In addition to the full length receptors, multiple
splice variants (SVs) of GnRHRs have been
identified in many vertebrate species. These variants
correspond to either shorter or longer transcripts.
For example, one SV from bullfrog (bf) GnRHR-
III is generated by exon skipping, whereas two
others arise from intron retention and a fourth from
transcriptional slippage (Wang et al., 2001). Alter-
native splicing was also reported in the mouse (Zhou
and Sealfon, 1994) and human GnRHRs (Kottler et
al., 1999) in which complete deletion of exon 2 was
observed. Similarly in chickens, multiple SVs have
been reported, and a SV of cGnRHR-I resulting in
the deletion of 90% of exon II (Sun et al., 2001 a),
and two SVs of cGnRHR-II (Figure 2) correspond-
ing to the deletion of exons 2/3 and part of exon 2
were identified (Shimizu and Bédécarrats, 2005).

Splice variants of the bfGnRHRs were shown to
be present in both the cell membrane and cytoplasm
of transfected HeLa cells, and to inhibit signalling of
the full length receptor, suggesting physical interac-
tions between receptors (Wang et al., 2001). Simi-
larly, a truncated human GnRHR-II consisting of
part of transmembrane domain 5 to the carboxy
terminus was shown to inhibit signaling of the full
length receptor in a dose-dependent manner when
coexpressed in COS-7 cells (Pawson et al., 2005).
The mechanisms by which these variants inhibit
signalling of the full length receptors are not known.
However, as suggested by Pawson et al. (2005), it is
possible that these SVs alter the normal de novo
synthesis of the full length receptors, possibly at the
level of the nucleus, endoplasmic reticulum, or
Golgi. It thus appears that an important relation-
ship exists between subcellular localization of SVs
and their inhibitory effect. Using fluorescence reso-
nance energy transfer, Cheung and Hearn (2005)
showed that both full length and SVs of wallaby
GnRHR form homo- as well as heterodimers. Fur-
thermore, microaggregation between GnRHRs is
promoted by GnRH agonist as a part of normal
receptor signal transduction (Cornea et al., 2001).
Although the functional relevance of SVs needs to
be elucidated further, these studies support the hy-
pothesis that SVs may be important in regulating
full-length receptor signal transduction.
<figure><img src="image_4.png" /><figcaption>Fig. 2. Schematic representation of the full length cGnRHR-II (panel A) and its splice variants (SV1 and SV2, panels B and C, respectively). Rectangular boxes represent cell plasma membrane. Each circle corresponds to an individual amino acid. Shaded amino acids correspond to sequences suspected of being important for receptor-ligand interaction (D and N in transmembrane domain 2), differential ligand selectivity (P-P-S in extracellular loop 3), and receptor activation (D-R-X-X-X-I/V-X-X-P-L in intracellular loop 2; D-P-X-X-Y in transmembrane domain 7). Single head arrows show cystein residues which may be involved in disulfide bond formation, whereas (*) show potential glycosylation sites. Double headed arrows correspond to the deletion site within splice variants.</figcaption></figure>
Distribution of GnRHRs and Control
of their Gene Expression

Most species with multiple GnRHRs possess at least one pituitary-specific form (Millar *et al.*, 2004). For example, in the bullfrog, three types of receptors were identified. *bfgnRHR-I* was shown to be pituitary-specific, whereas *bfgnRHR-II* and *III* are expressed in the forebrain and hindbrain (Wang *et al.*, 2001). Thus, *bfgnRHR-I* may play a role in the neuroendocrine control of gonadotropin secretion, and *bfgnRHR-II* and *III* may regulate other brain functions. In general, the “type-II GnRHRs” are widely distributed in the brain, as well as other tissues such as the pancreas, lung, and kidney (Millar, 2003). This is the case for *cGnRHR-I*, which is expressed in the brain, the pituitary, testes, spleen, and heart (Sun *et al.*, 2001a). In contrast, expression of *cGnRHR-II* was detected in the pituitary gland but not in brain tissues from both males and females (Shimizu and Bédécarrats, 2005). The SV of *cGnRHR-I* was also shown to be expressed in the same tissue as the full length receptor. Interestingly, mRNA encoding the longest variant of *cGnRHR-II* was predominantly detected in the pituitary, whereas mRNA from the shortest variant was detected in most brain tissues examined (Shimizu and Bédécarrats, 2005).

As mentioned previously, *in vivo* and *in vitro*
responsiveness to cGnRHs may depend on such
factors as sex and age. Interestingly, primary cul-
ture of chicken gonadotropes collected from juvenile
birds seem to better regain their responsiveness to
GnRH-I than cells harvested from adults (Liu *et al.*,
1996). However, the changes in Bmax and Kd
observed in pituitary cell membrane preparations
derived from hens at different ovulatory stages
(Kawashima *et al.*, 1992) suggest that gene expres-
sion of cGnRHRs and cell surface localization are
regulated in chicken pituitaries. In fact, an increase
in cGnRHR-I mRNA levels is observed in the pitu-
itary of hens throughout a 20 hour period preceding
ovulation, with maximum levels observed around
the time of the LH surge (Lovell *et al.*, 2005). We
did not observe any differences in cGnRHR-I
mRNA pituitary content between stages during a
reproductive cycle. However, we did observe dra-
matic changes in cGnRH-II mRNA levels (Shimizu
and Bédécarrats, 2005). In females, levels of

mRNA increase from the immature stage to the
peak of lay, then decreasing toward the end of the
reproductive cycle (Shimizu and Bédécarrats,
2005). Similarly, in males, levels of mRNA increase
from the immature stage up to 4 weeks post photo-
stimulation, decreasing thereafter (unpublished
data). It thus appears that cGnRHR-I and -II gene
expression are regulated by two different mecha-
nisms. Whether these two receptors are expressed in
the same pituitary cells is not known. However, it is
possible that cGnRHR-I is involved in the secretion
of LH during ovulatory cycles, while cGnRHR-II
plays a role in the development and maturation of
the reproductive axis post-photostimulation (Figure
1).

Molecular mechanisms involved in the control of mammalian GnRHR gene expression in various tissues have been extensively studied and were previously reviewed by Hapgood *et al.* (2005). Unlike their mammalian counterparts, the gene promoters of cGnRHRs have not been characterized and the mechanisms responsible for their expression remain to be determined. Nonetheless, castration of juvenile cockerels results in a significant increase in cGnRHR-I mRNA levels in the preoptic area and the anterior pituitary, and this increase is partially inhibited after treatment with oestrogen (Sun *et al.*, 2001a). Interestingly, no changes in cGnRH-I mRNA levels were observed in the mediobasal hypothalamus. Furthermore, treatment of intact adults with oestrogen did not affect levels of cGnRH-I (Sun *et al.*, 2001a). In non-laying hens, a single injection of ovarian steroid hormones (progesterone, estradiol or dihydrotestosterone) increases both the Kd and Bmax of pituitary cell membrane preparations (Kawashima *et al.*, 1992). Taken together, these results suggest that gonadal steroids have the ability to feed-back at the hypothalamus and pituitary level to control GnRHR gene expression. However, whether these steroids control both receptors the same way is questionable, and once again, the sex and reproductive status of the bird may be critical factors.

Activation of cGnRHRs and Initiation
of Intracellular Signalling

As members of the GPCR family, GnRHRs have
the ability to activate multiple intracellular second
messenger cascades depending on the type of G-
proteins involved. Coupling to $G_{αq}$ activates the phospholipase C pathway, resulting in the hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP₂) to produce inositol 1,4,5-triphosphate (IP₃) and diacylglycerol (DAG). In turn, IP₃ induces the release of calcium from endoplasmic stores, and DAG activates protein kinase C. Coupling to $G_{αs}$ or $G_{αi}$ activates and inhibits the adenylyl cyclase pathway, respectively, resulting in changes in cAMP production. In addition, binding of GnRH to its receptor can also activate L-type voltage sensitive calcium channels as well as non-voltage sensitive calcium channels (for review: Stojilkovic *et al.*, 1994; Millar *et al.*, 2004).

As expected, depletion of calcium in culture media or the use of calcium channel blockers reduces GnRH-induced release of LH from chicken pituitary cells (Davidson *et al.*, 1988; Liu *et al.*, 1995), and artificial activation of the PLC pathway increases the release of LH (Johnson and Tilly, 1991). These observations, coupled with the fact that stimulation of chicken pituitary cells with GnRH results in increased intracellular IP₃ levels (Davidson *et al.*, 1988), suggest association of cGnRHRs with $G_{αq}$. This was further confirmed after the cloning of both cGnRHR-I and -II (Sun *et al.*, 2001; Shimizu and Bédécarrats, 2005). However, whether cGnRHRs can also couple to other G-protein is not known. Interestingly, bfGnRHR-II couples more actively to $G_{αs}$ and bfGnRHR-I and -III couple to both $G_{αq/11}$ and $G_{αs}$ with similar efficiency, whereas rat GnRHR preferentially couples to $G_{αq/11}$, suggesting that differences in structure and/or in the amino acid sequence in intracellular loops may direct GnRHRs toward different signalling cascades (Oh *et al.*, 2003). It is worth mentioning that cGnRHR-II is 61, 51 and 65% homologous to bfGnRHR-I, -II and -III, respectively, whereas cGnRH-I has 51, 63 and 51% homology with the same receptors. It is thus possible that in chicken, as observed in the bullfrog, different GnRHRs possess different affinity for $G_{αq/11}$ and $G_{αs}$.

The relative affinity of cGnRHR-I and -II for the two chicken receptor types still needs to be determined. However, a high dose of mammalian GnRH or cGnRH-II stimulates IP₃ production to a greater extent in cells transfected with cGnRHR-II than cells transfected with cGnRHR-I (unpublished data). Type II GnRHRs have been shown to be

generally more sensitive to GnRH-II than to mammalian GnRH (Wang *et al.*, 2004), and in bullfrogs, bfGnRHR-I has a similar sensitivity to cGnRH II and mammalian GnRH whereas bfGnRHR-II and bfGnRHR-III show highest sensitivity for cGnRH-II and sGnRH, respectively (Wang *et al.*, 2001; Seong *et al.*, 2003). Likewise, cGnRHR-I has a higher affinity for cGnRH-II than mammalian GnRH, whereas the opposite is true for the human GnRHR (Sun *et al.*, 2001b). Interestingly, treatment of laying hens with buserelin (a mammalian GnRH super-agonist) does not substantially enhance the increase in LH observed with cGnRH-I or mammalian GnRH injection (Sterling and Sharp, 1984). In addition, some mammalian GnRHR antagonists behave as agonists to stimulate LH release from cultured chicken pituitary cells (Jacobs *et al.*, 1995), and some human GnRHR antagonists act as agonists on cGnRHR-I in transfected COS-1 cells (Sun *et al.*, 2001b). These experiments suggest that mammalian and chicken GnRHRs and GnRHRs possess structural requirements responsible for specificity. Sun *et al.* (2001b) suggested that $D$-Lys⁶ in mammalian antagonists interacts with the extracellular domain 2 of cGnRHR-I, and that this interaction is responsible for the agonist effect. Interaction between Arg⁸ of mammalian GnRH and an acidic residue in the extra-cellular loop 3 of mammalian receptors was proposed to be important for high affinity binding (Flanagan *et al.*, 1994). However, cGnRHR-I binds mammalian GnRH and $[Gln⁸]$ GnRH with the same high affinity, suggesting that alternative sites are probably involved in the binding of cGnRHR-I to its ligands (Sun *et al.*, 2001b).

Despite the difference in selectivity and affinity, several amino acid motifs important for ligand binding in other GnRHRs are also present in cGnRHRs (Figure 2). For example, Asp².⁶¹ (98) and Asn².⁶⁵ (102), which interact with the amino- and carboxy-terminal residues (pGlu-His, and Gly-NH₂, respectively) of GnRH (Davidson *et al.*, 1996; Flanagan *et al.*, 2000), are present in both cGnRHR-I and cGnRHR-II. A P-X-S/Y motif in extracellular loop 3 that was shown to be a determinant for differential ligand selectivity (Wang *et al.*, 2004) corresponds to a P-E-Y and P-P-S motif in cGnRHR-I and GnRHR-II, respectively. Furthermore, a $D$-RXXXI/VXXP-L motif in intracellular loop 2, and a DPXXY motif in transmembrane domain 7 that
are important for receptor activation (Ballesteros et al., 1998; Millar et al., 2004) are also present in cGnRHRs. However, the Lys<sup>3,32 (121)</sup> that was shown to be important for ligand binding in other receptors (Zhou et al., 1995) is present only in cGnRHR-I.

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