1 s2.0 S0378432005001302 Main

Animal Reproduction Science 88 (2005) 29–55
Synthesis and secretion of GnRH夽

Iain J. Clarke ∗ , Sueli Pompolo
Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton 3168, Australia
Abstract
Comprehensive studies have provided a clear understanding of the effects of gonadal steroids on
the secretion of gonadotropin releasing hormone (GnRH), but some inconsistent results exist with
regard to effects on synthesis. It is clear that regulation of both synthesis and the secretion of GnRH
are effected by neurotransmitter systems in the brain. Thus, steroid regulation of GnRH synthesis
and secretion can be direct, but the predominant effects are transmitted through steroid-responsive
neuronal systems in various parts of the brain. There is also emerging evidence of direct effects
on GnRH cells. Overriding effects on synthesis and secretion of GnRH can be observed during
aging, in undernutrition and under stressful situations; these involve various neuronal systems, which
may have serial or parallel effects on GnRH cells. The effect of aging is accompanied by changes in
GnRH synthesis, but comprehensive studies of synthesis during undernutrition and stress are less well
documented. Altered GnRH and gonadotropin secretion that occurs in seasonal breeding animals and
during the pubertal transition is not generally accompanied by changes in GnRH synthesis. Secretion of
GnRH from the brain is a reflection of the inherent function of GnRH cells and the inputs that integrate
all of the central regulatory elements. Ultimately, the pattern of secretion dictates the reproductive
status of the organism. In order to fully understand the central mechanisms that control reproduction,
more extensive studies are required on the neuronal circuitry that provides input to GnRH cells.
© 2005 Elsevier B.V. All rights reserved.
Keywords: GnRH; Gonadotropins; Reproduction; Neuroendocrinology; Estrogen
夽 This paper is part of the special issue entitled: GnRH in Domestic Animal Reproduction, Guest Edited by K.L.
Macmillan and Jin-Gui Gong.
∗ Corresponding author. Present address: Department of Physiology, PO Box 13F, Monash University, Clayton,
Vic. 3800, Australia. Tel.: +61 39594 4387; fax: +61 39594 6125.
E-mail address: iain.clarke@med.monash.edu.au (I.J. Clarke).
0378-4320/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2005.05.003
30 I.J. Clarke, S. Pompolo / Animal Reproduction Science 88 (2005) 29–55
1. Introduction
Gonadotropin releasing hormone (GnRH) is the brain factor that provides primary drive
for the reproductive axis. Without GnRH, the gonadotropes and the gonads do not function.
This is well demonstrated in the human condition of Kallman’s syndrome (Schwanzel-
Fukuda et al., 1989) and in the hypogonadal mouse (Charlton et al., 1983; Mason et al.,
1986). Given that GnRH secretion, and action on the pituitary gonadotropes, is essential to
the reproductive process, some knowledge of the factors that control the GnRH cells of the
brain is of fundamental importance. In spite of this, there is a relatively poor understanding
of the how various factors provide integrated information to the GnRH cells and regulate
secretion. The function of GnRH cells can be influenced by a range of factors, such as
gonadal feedback, stress, nutrition and season. This review focuses on how these intrinsic
and extrinsic factors regulate synthesis and secretion of GnRH.
Regarding the control of secretion, information is most abundant for studies done in
sheep, since there is a robust means for accessing the hypophyseal portal blood vessels in
this species (Clarke and Cummins, 1982). This allows minute-to-minute analysis of patterns
of secretion in detail and the changes that occur during the estrous cycle, and in response
to gonadal steroids, has been reviewed in detail (Clarke, 1996, 2002). Since the majority of
information on secretion of GnRH has been obtained from sheep, this review is focussed
on information from this species. In ovariectomised (OVX) females, the long-term effect of
both progesterone (P) and estrogen (E) is to inhibit GnRH secretion, whereas the transient
effect of unopposed elevation in E levels causes a positive feedback effect. In castrated
males, testosterone has a powerful negative effect on GnRH secretion. These steroid effects
can explain the patterns of luteinizing hormone (LH) secretion that are seen in gonad-
intact animals and explain the cyclic effects of LH secretion seen during the estrous cycle.
Information on the regulation of the synthesis of GnRH has been derived from various
species.
2. GnRH gene and amino acid sequence
GnRH is a 10 amino acid peptide that was first characterized in mammals (Amoss et
al., 1971; Matsuo et al., 1971). The mammalian gene, and the identification of a translated
precursor peptide was reported by Seeburg and Adelman (1984) and Adelman et al. (1986).
GnRH pre-prohormone mRNA was found in the diagonal band of Broca (dbB) and the
preoptic (POA) of the rat brain (Shivers et al., 1983). Using techniques such as immunohis-
tochemstry, in situ hybridisation, polymerase chain reaction and HPLC, expression of the
GnRH gene and the peptide product was detected in the brains of a wide variety of species
across the phylogenetic scale, from tunicates to humans (reviews in Fernald and White
1999; Lin et al., 1998b; Carolsfeld et al., 2000; Grove-Strawser et al., 2002; Gore, 2002a).
Sixteen different forms of GnRH have been isolated and in the majority of species, two or
more forms occur in anatomically and distinct neuronal populations (Gestrin et al., 1999;
Urbanski et al., 1999; Latimer et al., 2000). The amino acid sequence of the hypohysiotropic
GnRH is identical across mammals, with the exception of the guinea pig in which there are
substitutions of amino acids 2 and 7 (Jimenez-Linan et al., 1997). The gene sequence of
I.J. Clarke, S. Pompolo / Animal Reproduction Science 88 (2005) 29–55 31
the coding region and its molecular signal peptide sequence is highly conserved across a
wide range of species, differing only in a few amino acids (see Millar, 2003, this volume).
This chapter will focus on the cells of the mammalian brain that produce Type I GnRH and
provide the hypophysiotropic drive to the gonadotropes of the pituitary gland. GnRH-I is
found in the POA and hypothalamus, whereas cells that produce GnRH-II are found in the
midbrain and non-hypophyiotropic centres of the hypothalamus and hippocampus in mice,
rats, humans and monkeys (Miyamoto et al., 1982; White et al., 1995, 1998; Urbanski et
al., 1999; Gestrin et al., 1999). GnRH-III was first sequenced in salmon and is produced
in the olfactory and forebrain regions of several species of fish, reptiles and at least one
mammal, the capybara (Hydrochaeris hydrochaeris) (Sherwood et al., 1983; Montaner et
al., 1999; Carolsfeld et al., 2000). Using immunohistochemistry to study the sheep brain,
and using all of the currently available antibodies (Millam et al., 1989; Okuzawa et al.,
1990; Urbanski et al., 1999; Latimer et al., 2000), we have been unable to demonstrate the
presence of GnRH-II or GnRH-III in the cell bodies (Pompolo and Clarke, unpublished
data). Immunoreactive varicosities were seen in median eminence with these antibodies,
but this always co-localised with immunoreactivity for GnRH-I and could be blocked by
pre-absorption of the antibodies with GnRH-I (Pompolo and Clarke, unpublished data).
Some recent studies suggest that GnRH-III acts as an FSH-releasing factor (Yu et al., 1997,
2000; Dees et al., 2001; Densmore and Urbanski, 2003; Temple et al., 2003), but there is
no evidence that this peptide is secreted from the brain in mammals. We have been unable
to obtain evidence of secretion of GnRH-II into hypophyseal portal blood in sheep (G.A.
Lincoln, R.P. Millar and I.J. Clarke, unpublished data).
The gene encoding preproGnRH-1 gene has been cloned in number of species (reviewed
by Wierman et al., 1995; Lin et al., 1998; Fernald and White, 1999) and is approximately
4300-bp with four relatively short exons (1–4) separated by three large introns (A–C)
(Fig. 1). Exon 1 encodes the 5 -untranslated region, exon 2 the signal peptide, the GnRH-I
decapeptide and the first 11 amino acids of the GnRH-associated peptide (GAP). Exon 3
encodes amino acids 12–43 of GAP and exon 4 encodes the remainder of GAP and the
3 -untranslated region. In POA-AH of rat and human all three pro-GnRH-I introns are
spliced out of the primary gene transcript resulting in a mature mRNA of about 560 bases,
excluding the poly(A) tail (Adelman et al., 1986). Jakubowski and Roberts (1994) found
that the first processing step of prepro-GnRH-I in the POA-anterior hypothalamus of rats
Fig. 1. Structure of the GnRH-I gene that has been conserved throughout evolution (Lin et al., 1998). As a general
structure, the GnRH-I gene consists of four relatively short exons 1, 2, 3 and 4 (the rat gene has 138, 142, 96 and
186 bp, respectively). The exons are separated by 3 large introns A, B, C (the rate gene has 800, 1590 and 1350 bp
respectively). Black bars indicate 5 - and 3 -UTR in the exons 1 and 4 respectively. The hatched bar indicates
the signal sequence, the cross-hatched bar indicates the GnRH-I-coding region and the open bar indicates the
GnRH-associated peptide (GAP) coding region (Modified version of the sequence in H. burtoni from White and
Fernald 1998).
was the splicing out of intron B. Subsequently introns A and C are spliced out in no apparent
order. Other studies in the mouse and in GT1 cell lines suggested that there is no apparent
preferential order for the splicing out of introns (Yeo et al., 1996). In vitro studies, using
HeLa cells, show that the splicing of introns B and C occurs with greater efficiency than
that of intron A (Seong et al., 1999). More recent studies by Son et al. (2003) suggested
that the precise and efficient excision of intron A and the joining of adjacent exons is
probably the most critical regulatory step for the post-transcriptional regulation of GnRH
regulation. During the GnRH processing, several transcripts can be detected in the nucleus
of the cell including the unprocessed GnRH mRNA primary transcript, several processing
intermediates containing one or more introns, and the mature GnRH mRNA (Jakubowski
and Roberts, 1994; Yeo et al., 1996).
There are relatively few GnRH neurons scattered through the POA and adjacent areas,
with an estimated total population of 2400 GnRH cells in adult rhesus monkey brain
(Marshall and Goldsmith, 1980). Studies on transcriptional control of GnRH has been done
largely in GnRH-secreting cell lines such as GT1-7, GN10 and GN11 (Mellon et al., 1990;
Radovick et al., 1991). The 5 flanking region of GnRH gene is highly homologous between
species (Nelson et al., 1998). It comprises two major regions for transcription, an enhancer
and a promoter. A conserved 173-bp promoter is found just upstream of the transcriptional
start site and deoxyribonuclease I footprint analysis of the rat GnRH promoter revealed at
least seven regulatory regions located between −173 and 112 (Eraly and Mellon, 1995).
The promoter contains binding sites for transcription factors, such as POU-homeodomain
Oct-1 and the neuron-restricted homodomain protein, Otx2 (Kelley et al., 2000, 2002). The
proximal promoter also includes cis-acting elements involved in hormonal regulation by
glucocorticoids (Chandran and DeFranco, 1999), E and P (Kepa et al., 1996; Roy et al.,
1999) and 12-O-tetradecanoylphorbol 13-acetate (Bruder et al., 1996). A second important
region for transcription of the rat GnRH promoter is a 300-bp enhancer that is −1863 to
−1571 relative to the start site; this confers a 50–100-fold transcriptional activation over the
promoter alone, at least in GT1-7 cells (Nelson et al., 1998; Whyte et al., 1995). Within the
enhancer, specific binding sites for a number of transcription factors have been identified,
such as the zinc finger protein, GATA-4 and pbx-related protein (Lawson et al., 1996; Kelley
et al., 2002).
3. Anatomical localisation of GnRH in the brain
The GnRH neurons originate from the olfactory placode (Schwanzel-Fukuda and Pfaff,
1989; Wray et al., 1989; Wray, 2001). From this embryonic origin, the cells migrate to
and colonize the basal forebrain in and around the POA and the mediobasal hypothala-
mus (Wray, 2001, 2002). Studies in a number of species have shown a broadly similar
arrangement of GnRH localization in the brain, although there is some interspecific varia-
tion. Immunohistochemical (Baker et al., 1975; King et al., 1974; Polkowska et al., 1980;
Lehman et al., 1986) and radioimmunoassay (Wheaton et al., 1975) data show that the
median eminence of mammalian species contains the greatest amount of GnRH, this being
the area in which the peptide is stored in neuronal terminals prior to release into hypophyseal
portal blood. Significant amounts of GnRH are also found in the neuronal terminals of the
organum vasculosum of the lamina terminalis, although the function of these projections is
not known.
The GnRH neuronal cell bodies that project to the median eminence are mostly found in
the dbB, the POA and septal regions of the mammalian brain, although a discrete population
is found in the mediobasal hypothalamus; the importance of the latter is species dependent
(see Clarke, 1987b for detailed review). In the sheep, a small number of GnRH cells in the
arcuate nucleus may be relevant to basal secretion of peptide, with evidence of regulation
by ovarian steroids in the ewe (Boukhliq et al., 1999).
The anatomical arrangement between the hypothalamus, median eminence and the pitu-
itary gland is a masterpiece of design that allows for exquisite control of the gonadotropes
of the pituitary gland. The GnRH cells project to the external (secretory) zone of the median
eminence, where terminals are found in close proximity to the primary capillary bed of the
hypophyseal portal system (Page and Dovey-Hartman, 1984). Neural mechanisms regulate
the GnRH cells, although some intrinsic phasic mechanism probably exists within the cells
(Andrew and Dudek, 1983).
4. GnRH secretion
4.1. The relationship between GnRH and gonadotropin secretion
Original measurements of GnRH in plasma were made in anaesthetised rats using an

anaesthetic (Althesin® ) that does not inhibit secretion (Sarkar et al., 1976). With this
methodology, it was unequivocally shown that the signal for ovulation originated from the
brain. A pro-estrous surge in GnRH was demonstrated, inferring that this was the cause of
the preovulatory surge in LH secretion. Since original methods of collection of hypophyseal
portal blood compromised pituitary function (Worthington, 1966; Porter and Smith, 1967),
it was not possible to make simultaneous measurements of GnRH secretion and pituitary
gonadotrophin secretion. With the development of the ovine model (vide supra) to sample
hypophyseal portal blood in conscious animals and to simultaneously sample peripheral
blood, for the measurement of LH secretion (Clarke and Cummins, 1982), it became clear
that pulsatile pattern of LH secretion in OVX ewes is a direct reflection of hypothalamic
secretion of GnRH (although direct steroid feedback effects on the gonadotrope can
influence the amplitude of LH pulses). This was the final proof of the ‘neurohumoral
theory’ of Harris (1955), which stated that secretion of each type of hormone from the
anterior pituitary gland would reflect secretion of the relevant neuropeptide from the brain.
The system allows for substantial amplification of the signal, since picogram quantities
of GnRH in portal blood elicit pulses of LH in peripheral blood that are in the nanogram
range. Given the dilution in the periphery, this is an amplification of many 1000-fold. The
phasic pattern of GnRH and LH secretion allows for modulation of both frequency and
amplitude, and this is clearly manifest across the estrous cycle of the ewe (vide infra). The
high specificity of GnRH for the GnRH receptor on the pituitary gland confers specificity
to the whole reproductive process (see Nett, this volume). The low GnRH concentrations
in the hypophyseal portal blood and the presence of enzymes that degrade the peptide
(McDermott et al., 1981) necessitate that the sampling of portal blood is required in order
to monitor GnRH secretion. Interestingly, the pattern of secretion of follicle stimulating

hormone (FSH) is not tightly coupled to the secretion of GnRH, although GnRH action
is required for the maintenance of secretion of this gonadotropin (see review by Clarke,
1996). GnRH input to the gonadotrope appears to maintain FSH synthesis, but control of
the secretion of this gonadotropin seems to involve pituitary action of gonadal steroids and
inhibin (Clarke et al., 1984, 1986; Lincoln and Fraser, 1987; Li et al., 1989).
4.2. GnRH secretion during the estrous cycle
GnRH cells are regulated by the feedback effects of gonadal steroids, involving complex
processes that are not clearly understood. Fig. 2 shows the pattern of steroid secretion
across the estrous cycle of the ewe and indicates that GnRH cells are regulated by gonadal
steroids via intermediary neurons that possess the relevant steroid receptors. This defines
the pattern of GnRH secretion. GnRH neurons do not express P or androgen receptor
(Herbison et al., 1996; Skinner et al., 2001), nor do they express E receptor ␣ (ER␣)
(Shivers et al., 1983; Lehman and Karsch, 1993; Butler et al., 1999; Herbison and Pape,
2001). Although the GnRH cells of rat express ER␤ (Herbison and Pape, 2001; Hrabovszky
et al., 2000), the role of this sub-type of ER appears to be of minor significance with respect
to feedback control of GnRH cells (Krege et al., 1998; Lubahn et al., 1993). Lagrange et al.
(1995) showed that E hyperpolarised (and thus down-regulated) GnRH cells in the guinea
Fig. 2. Diagramatic version of the ovine estrous cycle showing the progesterone-dominant luteal phase, the
estrogen-dominant follicular phase and the surge-phase. Steroid hormones secreted by the ovary regulate the
GnRH cells via neuronal intermediates within the brain.
pig hypothalamus, by opening K+ channels. More recently, it has become increasingly

clear that steroids rapidly act on cells, in second or minutes, to exert effects that has been
classified as “nongenomic” (Falkenstein and Wehling, 2000; Razandi et al., 2003). Recent
work (Nunemaker et al., 2002) has utilised transgenic mice with green fluorescent protein
incorporated into the GnRH gene to allow identification of individual cells within the brain.
Using electrophysiological techniques these workers have reported direct effects of E on
GnRH cells. It was further suggested (Nunemaker et al., 2002) that such E effects are
modulated by afferents such as ionotropic GABA-ergic and glutamatergic neurotransmitter
systems. In spite of this, the overwhelming evidence is that the majority of control of GnRH
secretion is by steroids is via E-responsive systems within the brain, that relay peripheral
signals by conventional synaptic input to the cells. This indirect effect on controlling GnRH
secretion appears to involve both excitatory and inhibitory neurotransmitters/neuropeptides
(Herbison, 1998; Tilbrook et al., 2002).
The secretion of GnRH during the luteal phase of the cycle is characterised by high
amplitude, low frequency pulses (Moenter et al., 1991; Clarke, 1987a, 1995a, 1995b).
At the end of the luteal phase of the estrous cycle, with a fall in plasma P levels, the
frequency of GnRH and LH pulses increases (Moenter et al., 1991; Clarke, 1995b) (Fig. 3).
As the follicular phase ensues and plasma E levels rise (Fig. 2), GnRH pulse frequency
increases and amplitude decreases (Fig. 3). The preovulatory LH surge is associated with
an unambiguous rise in the secretion of GnRH (Clarke et al., 1987; Moenter et al., 1991)
(Fig. 3). This is due to the un-opposed action of E on the brain to activate systems that are
involved in the positive feedback mechanism (Clarke, 1995b). Thus, whilst under a negative
feedback clamp, high levels of E un-opposed by P causes activation of central mechanisms
that lead to positive feedback (Clarke, 1995b).
The estrous cycle may be modelled by appropriate treatment with E and P in OVX ewes
(Goodman et al., 1981; Evans et al., 1994a). This model appears to produce patterns of
GnRH/LH secretion similar to those seen during the normal cycle, indicating that these two
steroids play a predominant role in the regulation of GnRH cells in the female. In this model,
withdrawal of P and the application of higher levels of E lead to an artificial follicular phase,
followed by a preovulatory-like GnRH/LH surge. During the artificial follicular phase of
this model, GnRH secretion progressively declines, as in the normal cycle (Clarke, 1995a)
until the time that a GnRH/LH surge ensues (Evans et al., 1994b). This is a negative feedback
effect of E in the period prior to the surge and this is consistent with data obtained in cyclic
ewes (Moenter et al., 1991; Clarke, 1995b). With regard to the preovulatory LH surge, this
can be replicated by a single injection of estradiol benzoate to OVX ewes (Clarke, 1993)
or anestrous ewes (Clarke, 1988). Whilst this does not account for P priming of the brain,
that occurs in normal cyclic animals, the mere fact that a surge is produced indicates the
predominant role of E in the induction of the preovulatory event (Clarke, 1993).
P acts in two ways on the secretion of GnRH in the sheep. During the luteal phase, this
steroid limits GnRH pulse frequency (vide supra), whilst there is minimal effect at the level
of the pituitary gland (Clarke and Cummins, 1984). Continuously elevated plasma P levels
prevent the preovulatory LH surge (Scaramuzzi et al., 1971), which is due to blockade of
the GnRH surge (Kasa-Vubu et al., 1992). P also acts during the luteal phase of the cycle
to ‘prime’ the brain to the actions of E and this may affect the timing of the GnRH/LH
surge (Caraty and Skinner, 1999). Harris et al. (1998) treated P-primed OVX ewes with E
Fig. 3. GnRH and LH levels at various stages of the ovine estrous cycle. During the progesterone-dominant
luteal phase (A), GnRH and LH pulses are of low frequency and high amplitude. During the follicular phase, the
frequency of GnRH/LH pulses increases and the amplitude decreases; there is a progressive decline in amplitude
from the early follicular phase (24 h after an injection of Cloprostenol® to late-luteal phase ewes) (B) until the late
follicular phase (48 h after an injection of Cloprostenol® to late-luteal phase ewes) (C). There is an unambiguous
surge in GnRH secretion that initiates the preovulatory LH surge and ovulation (D), in concert with synonymous
upregulation of the sensitivity of the pituitary gonadotropes to GnRH. Arrowheads in panels A–C indicate defined
pulses. In the upper part of panel D, the larger data points represent estradiol levels in peripheral plasma and
the smaller data points represent GnRH levels in hypophysial portal plasma; the data in the lower part represent
peripheral plasma levels of LH. Panels A–C are adapted from Clarke (1995a) and panel D is adapted from Moenter
et al. (1991).
for 8 h and then gave P for 5 h afterwards or between 5 and 10 h afterwards. Only when P
was given in the period shortly after E treatment was the LH surge blocked.
E appears to activate cells within the mediobasal hypothalamus to cause the GnRH/LH
surge (Caraty et al., 1998). Cells of the arcuate nucleus and the ventromedial nucleus, and
in the A1 field of the brainstem of the OVX ewe are activated by E, as indicated by the
appearance of Fos within 1 h of E injection (Clarke, 1995b; Clarke et al., 2001; Rawson
et al., 2001). The E-responsive cells of the brainstem are retrogradely labelled from the
preoptic area (Rawson et al., 2001) are noradrenergic and contain ER␣ (Scott et al., 1999).
In the arcuate/ventromedial region of the hypothalamus the types of cells that express ER␣
and show Fos responses to E were not well characterised (Clarke et al., 2001), but recent
data (Pompolo et al., 2003b) show that many of these are glutamatergic. It appears that there
is an indirect pathway from this region of the hypothalamus to GnRH cells (Pompolo et
al., 2001), but this is not fully defined. The glutamatergic cells that are activated by E may
form part of this pathway (Pompolo et al., 2003b). Although induction of cells by E can be
detected in the relevant regions, and it is possible that these are related to control of GnRH
synthesis/secretion, it is also possible that these elements are involved in other functions
such as sexual behaviour.
4.3. GnRH secretion in rams
Tilbrook et al. (2002) have reviewed studies carried out in rams. The relationship between
GnRH and LH secretion is demonstrated in this sex, as in the female. Testosterone negatively
regulates the secretion of GnRH in castrated rams, but gonadal steroids do not appear to
regulate the gonadotropes directly in rams (Tilbrook et al., 1991). Inhibin clearly acts to
regulate FSH secretion at the level of the pituitary gland in rams (Tilbrook et al., 1993,
1999, 2001).
5. GnRH synthesis
5.1. Effects of gonadectomy and gonadal steroids on GnRH synthesis in females
The rate of production of mature GnRH peptide can potentially be influenced by

transcription rate, mRNA stability, and post-translational processing (Gore and Roberts,
1997), but most studies have involved measurement of steady-state levels of mRNA,
using techniques such as in situ hybridisation. There have, however, been some studies
of regulation of transcription, as indicated below. Results obtained in animals of various
species, examining levels of mRNA expression across the normal estrous cycle, and
following castration, have not always produced consistent results, so the exact role of
gonadal steroids in the regulation GnRH synthesis remains controversial (see reviews
by Sagrillo et al., 1996; Gore and Roberts, 1997; Gore, 2002a). Expression during the
normal cycle was found to be inversely proportional to plasma levels of oestradiol in rats,
suggesting negative feedback regulation (Zoeller and Young, 1988; Park et al., 1990),
but at least one study reported no cyclic changes in the levels of GnRH mRNA in rats
(Malik et al., 1991). Using in situ hybridisation, one group (Porkka-Heiskanen et al., 1994)
observed an increase in the number of cells expressing GnRH mRNA at 1200 h on the day
of pro-estrus, but the level of mRNA expression/cell was not altered. Similar results were
observed in rats (Park et al., 1990), but the increase was seen at 1800 h. Gore and Roberts
(1995) used a ribonuclease protection assay and observed an increase in primary transcripts
of GnRH and mature message at 1500 h. Others (Suzuki et al., 1995) have used quantitative
reverse transcription-polymerase chain reaction (PCR) methodology and observed a peak
of expression at 1100 h on the day of pro-estrus. Thus, an increase in GnRH mRNA
expression and/or the number of cells in which GnRH expression appears to occur on the
day of proestrus, but the timing of this increase can vary. Perhaps such differences relate to
subtle differences in the timing of the preovulatory GnRH/LH surge in different colonies
of rats.
Early studies using radioimmunoassay showed that ovariectomy reduced levels of GnRH
in the mediobasal hypothalamus in rats (Kalra, 1976) and sheep (Wheaton, 1979), primarily
due to the reduction in content of GnRH in the median eminence (Kalra, 1976; Kobayashi
et al., 1978) and this effect could be reversed in female rats by administration of E (Kalra,
1976). There appeared to be very little, if any, change in the GnRH content of hypothalamic
nuclei of rats following ovariectomy (Kobayashi et al., 1978). Using different techniques,
some studies showed that GnRH mRNA levels were increased in OVX rats compared to
randomly cycling female rats (Toranzo et al., 1989), whereas others reported no change
compared with intact pro-estrous rats (Kelly et al., 1989), or a reduction of expression
compared to prepubertal rats (Kim et al., 1989) or diestrous rats (Roberts et al., 1989). These
differing results are probably due to several factors, such as different models, the length of
time after the ovariectomy, or time of the day that animals were killed, or in which stage of
estrous cycle these animals were killed. Roberts et al. (1989) showed that GnRH expression
was lower 9 days after ovariectomy than at shorter (2 days) or longer (16 and 24 days)
time-points when compared with randomly cycling rats and Petersen et al. (1993) observed
that GnRH mRNA decreased between 2 and 28 days after ovariectomy. Others (Kim et
al., 1989) observed reduced levels of GnRH mRNA within 2 days after ovariectomy of
immature, age-matched rats. Toranzo et al. (1989) observed an increase of 25% in the levels
of GnRH mRNA when compared with randomly intact animals. On the other hand, Kelly
et al. (1989) did not find any effect 14 days after OVX when compared with proestrous
rats.
E replacement in OVX rats induces both negative and positive feedback effect on LH
secretion. On one hand, E treatment reverses the rise in basal plasma LH levels following
ovariectomy but also stimulates a daily afternoon LH surges (Legan et al., 1975). Levels of
GnRH expression are lower on the morning of the surge but increase in the afternoon when
the surge occurs (Zoeller et al., 1988; Petersen and Barraclough, 1989; Toranzo et al., 1989;
Rosie et al., 1990; Weesner et al., 1993a; Petersen et al., 1993, 1995, 1996).
In early studies in sheep, Wheaton (1979) using radioimmunoassay, was unable to reverse
the effect of ovariectomy by administering E, which is somewhat puzzling given the con-
sistent responses achieved in rats. More recent work, using in situ hybridisation, showed a
decrease in GnRH mRNA levels prior to an E-induced LH surge in OVX ewes (Dhillon et
al., 1997; Harris et al., 1998). There have, however, been no comprehensive studies of the
changes in GnRH mRNA levels across the estrous cycle in the ewe. Thus, studies in the rat
show that mRNA levels increase prior to the LH surge, whereas studies in the sheep show
the opposite. In OVX ewes given a low continuous dose of E, P treatment does not affect
the level of GnRH mRNA/cell, as indicated by in situ hybridisation studies (Robinson et al.,
2000). In the OVX female monkey treated with either E or E + P, both treatments reduced
GnRH mRNA levels, but there was no difference between E or E + P treatments compared
to levels in untreated OVX animals (Krajewski et al., 2003). These data suggest that the
action of P to reduce GnRH pulse frequency during the luteal phase of the cycle is not
accompanied by an effect on the synthesis of GnRH. In the rat, the situation is somewhat
more complicated, because P can cause a surge in GnRH secretion in the E-primed, OVX
female (this does not occur in species such as the sheep). Such treatment advances the
LH surge, when compared to treatment with E alone (DePaolo and Barraclough, 1979).
In OVX-E treated prepubertal female rats, a single injection of P (to induce an LH surge)
causes an increase in GnRH mRNA expression prior to the induced LH surge (Kim et al.,
1989). On the other hand, administration of P markedly potentiated the negative feedback
effect of E on pro-GnRH gene expression in OVX animals treated for 14 days (Toranzo et
al., 1989). This demonstrates the duality of action of P on the GnRH/LH system in rats.
5.2. Effects of gonadectomy and gonadal steroids on GnRH synthesis in males
Early studies on the effects of gonadectomy and gonadal steroid replacement on GnRH
secretion in vivo and in vitro in the rat, has been extensively reviewed by Kalra and Kalra
(1989). Wiemann et al. (1990) observed that the number of cells that expressed GnRH
mRNA was lower in intact male rats compared to those castrated for 21 days, but did see a
difference in the level of expression/cell.
Selmanoff et al. (1991) also used in situ hybridisation and found that castration of male
rats caused a significant increase in GnRH mRNA in the rostral hypothalamus of animals
killed 9 days after castration. Another series of studies show that gonadectomy caused
an increase of 54% in the level of expression of GnRH mRNA/cell in male rats14 days
after gonadectomy, but this effect could be reversed by the 5␣-reduced derivative of testos-
terone, dihydrotestosterone (Li et al., 1995). In contrast to the above, one group (Spratt and
Herbison, 1997) found that GnRH mRNA expression in long-term castrated rats was
increased by either testosterone or E treatment. Thus, effects of steroids on GnRH expres-
sion (at least in rats) are not consistent. In castrated male sheep, testosterone treatment had
no effect on the level of GnRH mRNA expression (Hileman et al., 1996), suggesting that
the effect of testosterone to reduce secretion (vide supra) is not accompanied by an effect
on synthesis.
5.3. Neurotransmitter effects on GnRH synthesis
Whilst gonadal steroids such as E and P are the cyclic regulators of GnRH secretion,
these are not considered to have direct effects on GnRH neurons (vide supra). It follows,
therefore, that effects of steroids on GnRH synthesis must involve neuronal intermediaries
(Fig. 2). GnRH cells receive input from various neuronal systems (Tilbrook et al., 2002).
Studies in rats have shown that for some neurotransmitters, inhibitory effects can be seen in
OVX animals, but stimulatory effects are seen in steroid treated-OVX animals. Such a dual
effect can be seen with adrenergic agonists and neuropeptide Y (Kalra and Kalra, 1983). For
this reason, the experimental model in which effects of drugs are being examined should be
carefully noted. The following is a review of our current understanding of which systems
may regulate GnRH synthesis.
GABA is thought to be an inhibitor of GnRH secretion (Herbison, 1998) and treatment
of male rats with a GABAA agonist or benzodiazepine receptor ligand causes a decrease
of GnRH mRNA, as measured by in situ hybridization (Li and Pelletier, 1993a, 1995a).
Similar results are obtained in OVX female rats treated with a GABAA agonist (Leonhardt
et al., 1995). On the other hand, Bergen et al. (1991) found that treatment of OVX rats
with bicuculline (GABAA antagonist) decreased GnRH mRNA expression and Kang et
al. (1995) showed an increase in GnRH mRNA in OVX-E + P-treated rats treated with the
GABAA agonist muscimol. These latter data suggest a stimulatory role for the GABAergic
system in the control of GnRH synthesis, so the effect of GABA on GnRH mRNA levels is
not clear. Grattan et al. (1996) have presented evidence to suggest that the feedback effect
of testosterone on GnRH neurons is mediated by androgen-sensitive GABA neurons in the
preoptic region of the male rat brain.
Serotonin stimulates LH release in presence of high levels of E (such as occur around the
preovulatory surge), but inhibits LH release in OVX rats (Vitale and Chiocchio, 1993). Work
in intact male rats (Li and Pelletier, 1995b) showed that serotonin reduces GnRH mRNA
levels. Although GnRH mRNA levels were increased by the 5-HT2 receptor antagonist, this
was not counter-acted by concomitant treatment with a 5-HT3 antagonist (Ondansetron).
The latter authors suggested the serotoninergic system exerts a negative feedback effect on
the biosynthesis of GnRH via activation of 5-HT2 receptor. To our knowledge, the effect
of serotonin on GnRH mRNA expression in females is not known.
Dopamine appears to be an important regulator of GnRH cells in rodent species (Kalra
and Kalra, 1983; Li and Pelletier, 1992a, 1992b; Kalra, 1993). In ewes, pharmacological
studies have implicated dopamine as an important neurotransmitter mediating the negative
feed back effect of E on GnRH/LH secretion during the anestrous season (Le Corre and
Chemineau, 1993; Meyer and Goodman, 1985). Whereas the feedback effects of E in ewes
clearly involve D2 receptors (Anderson et al., 2001), this does not appear to be the case
with testosterone feedback in males (Tilbrook and Clarke, 1992). There are no studies on
dopaminergic regulation of GnRH expression in this species. In rodents, dopamine can
exert a stimulatory (Choudhury et al., 1974; Clemens et al., 1977; Vijayan and McCann,
1978a, 1978b) or inhibitory effect when on LH secretion (Drouva and Gallo, 1977; Beck
et al., 1978; Judd et al., 1978; Sarkar and Fink, 1981). Treatment for 14 days with a D2
receptor agonist increased GnRH mRNA expression in female and male rats by 32 and 67%,
respectively, while the dopamine receptor antagonist haloperidol had the opposite effect.
Nitric oxide (NO) is a diffusible gas that is formed in vivo by conversion of the amino
acid l-arginine to l-citrulline, the reaction being catalysed by nitric oxide synthase (NOS).
NO acts as neurotransmitter in the brain and stimulates LH release (Bonavera et al., 1993;
Ceccatelli, 1997). Consistent with this, the infusion of NOS inhibitors into the lateral ven-
tricles reduces GnRH mRNA levels in adult intact and castrated male rats (Wang et al.,
1998).
Noradrenaline stimulates LH secretion in steroid-primed female rats, but reduces
LH secretion in OVX females (Kalra, 1993). The stimulatory effect appears to be via
the ␣1 adrenergic receptor (Herbison, 1997). An increase in GnRH mRNA expression
in OVLT/POA region has been observed, using in situ hybridisation, within 1 h of

intraventricular infusion of noradrenaline to OVX-E treated rats (He et al., 1993). Infusion
of Prazosin (␣1 antagonist) into the POA of OVX-E treated rats reduces GnRH expression
at the time of the E-induced LH surge (Weesner et al., 1993b), consistent with a stimulatory
effect of noradrenaline at this time. Kim et al. (1994) treated OVX + E rats with an inhibitor
of dopamine ␤ hydroxylase (diethyldithiocarbamic acid) 1 h before treatment with P and
a decrease in GnRH mRNA expression was observed using Northern blot analysis. In
the same animal model, these workers (Kang et al., 1998) showed that a noradrenergic
neurotoxin (N-(2-chorethyl)-N-ethyl-2-bromobenzylamine) also reduced GnRH mRNA
levels in the POA. The authors suggested that suppression of the noradrenergic transmission
resulted in a marked decrease in P-induced GnRH levels. Thus, the literature consistently
shows that noradrenergic input provides a stimulus to GnRH synthesis.
Like noradrenaline, NPY may exert dual effects on GnRH/LH secretion in rats depending
upon the steroid status (Kalra, 1993). In a singular report (Li et al., 1994), intracerebroven-
tricular infusion of NPY or a Y1 receptor agonist to castrated adult male rats was found to
stimulate the level of GnRH expression, as measured by in situ hybridisation.
Opioids have an inhibitory effect on GnRH release and are thought to be one means
by which negative tone is imposed on GnRH secretion (Gore and Terasawa, 2001; He et
al., 1993; Kalra, 1993). In male rats, chronic morphine treatment reduced GnRH mRNA
expression whereas naloxone (a broad acting antagonist), increased expression (Li and
Pelletier, 1993b). Short-term (15 min) morphine treatment of female rats (of unspecified
reproductive state) did not alter steady-state levels of mRNA (He et al., 1993), but when
this treatment was followed by intraventricular infusion of noradrenaline, this blocked the
NE-induced increase in GnRH mRNA. This inhibitory effect of opioids on the expression
of GnRH mRNA may mediate the negative feedback effects of steroids on GnRH release
through an inhibition of biosynthesis; this may involve counter-action of the stimulatory
actions of adrenergic input.
The amino acid glutamate is implicated in direct and indirect regulation of GnRH/LH
secretion (Dhandapani and Brann, 2000). N-Methyl-d-aspartate (NMDA) receptor subtypes
and kainate receptors are found on GnRH neurons and glutamatergic inputs to GnRH neu-
rons have been demonstrated in several species (Goldsmith et al., 1994; Eyigor and Jennes,
2000; Gore, 2001, 2002b; Lin et al., 2003; Ottem et al., 2002; Pompolo et al., 2003a,b).
Using a quantitative RNase protection assay, and in randomly cycling female rats, it was
shown that N-methyl-d,l-aspartate and kainic acid effected a rapid (within 1 h) increase
in GnRH mRNA expression (Gore and Roberts, 1994). A similarly rapid effect is seen
with NMDA infusion to intact male rats (Petersen et al., 1991) and in androgen-sterilised
female rats. Intravenous infusion of NMDA into OVX E + P-treated rats doubled the levels
of GnRH mRNA levels in medially located neurons (determined by in situ hybridisation),
but this was not blocked by MK801 (NMDA receptor antagonist) (Ottem and Petersen,
2002). Others have shown a decrease in GnRH mRNA after treatment with MK801 using
Northern blot analysis (Seong et al., 1993), PCR (Suzuki et al., 1995) and RNase pro-
tection assays (Gore and Roberts, 1997). Using in situ hybridization, and in mice of both
sexes, NMDA increased GnRH mRNA expression and the antagonist CGP40116 reduced
expression (Ford and Ebling, 2000). On the other hand, a study in male mice with use
of a ribonuclease protection assay (Wu et al., 2000) showed that NMDA caused a rapid
(within 15 min) decrease in cytoplasmic GnRH mRNA with a reduction in nuclear GnRH
primary transcript RNA after 2 h. In neonatal mice (Ford and Ebling, 2000) the glutamate
antagonist CGP 40116 did not alter GnRH mRNA expression at on Day 1 of postnatal life,
but a reduction was observed at Day 5. Others (Liaw and Barraclough, 1993) found that
treatment with NMDA increased GnRH mRNA expression on the day of birth, but not on
Day 5. In summary, glutamatergic input appears stimulatory to GnRH synthesis.
6. Changes in GnRH secretion and synthesis
6.1. Effects of puberty and aging
Hypothalamic levels of GnRH content and release increase steadily during postnatal
development (Sisk et al., 2001; Plant and Shahab, 2002) in rats and monkeys. A study of
mice (Ford and Ebling, 2000) examined levels of GnRH mRNA across prenatal and post-
natal life and during the sexual maturation of males and females using in situ hybridization.
Expression increased between embryonic Day 17 and postnatal Day 10, with no sex dif-
ference. Using their RNase protection assay to study mice, Gore et al. (1999) also found
that cytoplasmic GnRH mRNA levels increased gradually during post-natal development
in both sexes. Levels of GnRH primary transcript (an index of GnRH gene transcription)
showed a four-fold increase between postnatal Days 5 and 7 and reached adult levels at
post-natal Day15 in males. In females the levels of GnRH primary transcript were high at
embryonic Day 16, decreased to nadir at postnatal Day 5, and then increased at postnatal
Day 7; levels were then stabilised and did not increase further in adulthood (Gore et al.,
1999). In one study, using in situ hybridization , no difference in GnRH expression was
found between pre-pubertal and adult male rats (Wiemann et al., 1989). In contrast, using the
RNase protection assay, GnRH mRNA expression in the brains of rats was found to increase
at postnatal Day 30 in females, followed by a further increase to adult levels at postnatal Day
45 (Jakubowski et al., 1991). Similar trends were observed in male rats, with an increase in
expression between postnatal Days 22 and 26, followed a further increase at postnatal Day
40 (Dutlow et al., 1992). Levels of GnRH mRNA expression are greater in post-pubertal
(60-day-old) hamsters than in pre-pubertal (21-day-old) hamsters, when measured by in
situ hybridization (Parfitt et al., 1999). The apparent increase in GnRH secretion at puberty
could reflect a change in the activity of GnRH neurons or central mechanisms controlling
secretion. A study in the male rhesus monkey examined animals castrated at the age of 1
week and killed at the 4–7 weeks (neonates) or 12–15 months (juvenile) (El Majdoubi et al.,
2000). This showed that GnRH mRNA levels were similar in both instances, but the study
did not include post-pubertal animals. Thus, the weight of evidence from various species is
that GnRH synthesis rises during development to reach adult rates prior to puberty; puberty
is not due to a sudden increase in the rate of production of GnRH.
Aging is characterized by attenuation of pulsatile secretion of LH release and reduced
size of the preovulatory GnRH/LH surge (Wise et al., 2002). Rodents have been well
studied during the aging process, which is characterized by irregularity in estrous cyclicity
and persistent estrus in females. This transition to acyclicity occurs independent of ovarian
follicular atresia, since resumption of ovulation may occur in response to external stimuli
(Lu et al., 1977; Huang et al., 1978). In rats, attenuation or delay in the preovulatory LH
surge occurs in middle-aged females. In intact female rats and humans, GnRH mRNA levels
increase with the aging process (Rance and Uswandi, 1996; Gore et al., 2000) presumably
because of upregulation of expression due to lack of steroid feedback. On the other hand,
GnRH gene expression is reduced in OVX female rats, indicating an overall reduction in
the capacity of these cells to synthesise GnRH (Rubin et al., 1997; Miller and Gore, 2002).
In contrast, GnRH mRNA levels are reduced with aging in intact male rats (Gruenewald
et al., 2000). In summary, the increase in GnRH mRNA expression with aging in females
is consistent with the loss of ovarian cyclicity and the removal of negative feedback of
ovarian steroids on the GnRH cells of the brain; this is not seen on OVX animals because
there is no negative tone. The anomalous result in males is not readily explicable, although
sexual dimorphism of the steroid regulation of GnRH synthesis has been recently reported
(Thanky et al., 2003).
6.2. Effects of season/photoperiod
Some species, such as sheep and hamsters, are seasonal breeders, utilising photoperiod
and other cues to regulate their circannual pattern of reproduction (Thiery et al., 2002). An
extensive literature exists on the seasonal patterns of LH secretion (reflecting GnRH secre-
tion) (Karsch et al., 1993; Lehman et al., 2002; Lincoln, 2002), but this is beyond the scope
of the present review. The following is confined to seasonal effects on synthesis, which
is less extensively reviewed in existing literature. Siberian hamsters breed under long-day
photoperiod. In males of this species (intact or castrated with testosterone replacement), the
expression of GnRH mRNA in the dbB/septal area was higher in short-day photoperiod than
in long days (Ronchi et al., 1992). Thus, synthesis appeared to be increased during the non-
breeding period. Others (Brown et al., 2001) have observed no difference in mRNA levels
using in situ hybridisation in intact male hamsters on either short or long-day photoperiods.
Neither was any difference observed in gray hamsters (Kawamoto et al., 2000) on short-
or long-day photoperiod. In another experimental series, hamsters were transferred from
short (6L:18D for 4 weeks) to long-day photoperiod (16L:8D for 1 day) (Porkka-Heiskanen
et al., 1997) and a transient increase was observed in the number of cells expressing GnRH
mRNA. In a study of male prairie were housed under short or long days and at two different
temperatures (mild-20 ◦ C or low-8 ◦ C), no difference in the level of GnRH mRNA expres-
sion was observed when measured by in situ hybridisation (Kriegsfeld et al., 2000). In cas-
trated male sheep with or without testosterone replacement and kept for 3 weeks in short or
long-day photoperiod, no difference was observed in GnRH mRNA levels although plasma
concentrations of LH were lower under long days (Hileman et al., 1998). Thus, the weight of
evidence suggests that seasonal breeding status is not the result of altered synthesis of GnRH.
6.3. Nutritional effects
Food deprivation of sheep reduces plasma LH levels (Campbell et al., 1977; Henry et al.,
2001) as result of inhibitory influences on secretion of GnRH (Thomas et al., 1990; I’Anson
et al., 2000; Henry et al., 2001; Henry, 2003). In situ hybridisation studies by McShane et
al. (1993) showed that the levels of GnRH mRNA were not altered in OVX lambs following
30% food-restriction for 7 weeks. Similar results have been obtained with food restriction
of intact male rats for 5 (Bergendahl et al., 1992) or 17 days (Leonhardt et al., 1999). Oth-
ers (Gruenewald and Matsumoto, 1993) have observed a decrease in the number of cells
expressing GnRH mRNA in the dbB/mPOA of male rats subjected to food restriction for
3 months, while the level of expression/cell was unchanged. In male prairie voles, food
restriction led to an increase in the number of GnRH cells detected by immunohistochem-
istry with an increase in cell size and concomitant increase in GnRH-immunoreactive fibre
intensity the median eminence (Kriegsfeld et al., 2001); these authors suggested the this
was probably a reflection of reduced secretion of GnRH. Interestingly, treatment of under-
nourished animals with leptin can correct the hypogonadotropic stated (Henry et al., 2001),
suggesting that this fat-derived hormone relays important information to the brain regarding
metabolic status and this is ‘sensed’ by pathways regulating reproduction.
6.4. Effects of stress
It is well recognised that stress has deleterious effects on the reproductive axis (Tilbrook
et al., 2002). Whilst many studies show that this is manifest in a reduction in gonadotropin
secretion, few show direct effects on the GnRH system. Recent studies, Debus et al. (2002)
showed that administration of a bacterial endotoxin, to induce immune/inflammatory stress,
reduced pulsatile GnRH/LH secretion; the effect on GnRH synthesis was not measured.
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Animal Reproduction Science 88 (2005) 29–55

Synthesis and secretion of GnRH夽

Keywords: GnRH; Gonadotropins; Reproduction; Neuroendocrinology; Estrogen

2. GnRH gene and amino acid sequence

3. Anatomical localisation of GnRH in the brain

4.1. The relationship between GnRH and gonadotropin secretion

Original measurements of GnRH in plasma were made in anaesthetised rats using an

to monitor GnRH secretion. Interestingly, the pattern of secretion of follicle stimulating

4.2. GnRH secretion during the estrous cycle

pig hypothalamus, by opening K+ channels. More recently, it has become increasingly

4.3. GnRH secretion in rams

5.1. Effects of gonadectomy and gonadal steroids on GnRH synthesis in females

The rate of production of mature GnRH peptide can potentially be influenced by

5.2. Effects of gonadectomy and gonadal steroids on GnRH synthesis in males

5.3. Neurotransmitter effects on GnRH synthesis

in OVLT/POA region has been observed, using in situ hybridisation, within 1 h of

6. Changes in GnRH secretion and synthesis

6.1. Effects of puberty and aging

6.2. Effects of season/photoperiod

6.3. Nutritional effects

6.4. Effects of stress

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