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Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge CB2 4AT, United Kingdom
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Abstract |
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 Top Abstract I. IntroductionII. Effects of Estrogen...III. Brain Sites of...IV. Pathways for Estrogen...V. Model of Estrogen...VI. Concluding RemarksReferences |
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I. Introduction |
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TopAbstractI. IntroductionII. Effects of Estrogen...III. Brain Sites of...IV. Pathways for Estrogen...V. Model of Estrogen...VI. Concluding RemarksReferences |
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Estrogen is one of the principal determinants of GnRH neuron functioning and, acting as a classic homeostatic feedback molecule between gonad and brain, is critical in enabling these cells to exhibit fluctuating patterns of biosynthetic and secretory activity. For the greater part of the ovarian cycle, estrogen helps restrain LH secretion through what has been termed its "negative feedback" action. This has been shown to occur, in part, through an inhibition of GnRH secretion in several species (7, 11, 12, 13), but also involves potent actions of estrogen on the pituitary gonadotrophs (3, 4, 14). Estrogen also exhibits a "positive feedback" influence upon the GnRH neurons and pituitary gonadotrophs to generate the preovulatory LH surge. Whereas the rising follicular phase concentrations of circulating estradiol are sufficient on their own to generate the GnRH surge in some spontaneously ovulating species (8, 10, 15), others, such as the rat, require the rising levels of estrogen to coincide with a circadian input to trigger the surge (11, 16, 17). Together, these observations indicate an important, indeed critical, influence of estrogen upon the functioning of mammalian GnRH neurons.
The purpose of this review is to provide an account of the effects of estrogen upon the GnRH neuron and describe how this may come about. The latter issue is not simple as these cells are not thought to express classic nuclear estrogen receptors (ERs) (see below). It is important at the outset to state the intended limitations of the review. First, it focuses entirely upon the female and the cyclical patterns of GnRH neuronal activity encountered in the reproductively active adult. Data from rodent, ovine, and nonhuman primate species are considered. Second, although it is recognized that estrogen exerts important physiological actions at the level of the pituitary gland (4, 5, 14), the present review focuses specifically upon the central mechanisms of estrogen action. To this end, GnRH secretion data have been sought and used extensively but, where unavailable, inference has been taken with due care from LH measurements. Third, because of the importance and potential similarities in estrogen’s influence upon the GnRH neuron across species, the review does not deal with progesterone actions. The one exception will be in regard to the rat where, as in the human (18), progesterone has a role in the generation of the normal shape of the estrogen-induced LH surge (19). This influence is entirely dependent upon estrogen preexposure (20) and, where possible and appropriate, the interactions between estrogen and progesterone are highlighted in the rat.
Finally, a definition. The "GnRH pulse generator" and "GnRH surge generator" are phrases that were used extensively in the past to refer to the ill-defined neural elements responsible for the pulsatile and surge patterns of GnRH secretion, respectively. While such constructs are helpful in a conceptual sense, they are difficult in that they imply the unproven hypothesis that specific "pulse" and "surge" GnRH neuronal systems exist. Hence, for the purposes of this review, I will refer to the "GnRH network." This is defined as "the GnRH neurons, the glial cells directly associated with them, and the non-GnRH neuronal populations involved in regulating the activity of GnRH neurons." Using these definitions and focus, I intend to 1) provide a catalog of the effects of estrogen on the GnRH neuron, 2) highlight three different modes of estrogen action within the GnRH network, and 3) propose a simple framework model for how estrogen may engender cyclical activity upon the GnRH neurons.
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II. Effects of Estrogen on GnRH Neurons |
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TopAbstractI. IntroductionII. Effects of Estrogen...III. Brain Sites of...IV. Pathways for Estrogen...V. Model of Estrogen...VI. Concluding RemarksReferences |
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The precise developmental period during which estrogen, derived from testosterone, exerts its "organizational" influence upon the GnRH network varies among species. In the rat, this so-called "critical period" for masculinization of the GnRH network is near the time of birth. Testosterone or estrogen administered to female rats on single or multiple days over the first 5 postnatal days will render them unable to generate an LH surge after puberty (23, 26, 32) whereas the gonadectomy of male rats must occur within a few hours of birth if they are to exhibit female-like LH surges as adults (26, 39). In the sheep, the critical period for testosterone exposure is relatively prolonged and occurs between gestational days 30–100 with masculinization of the LH surge mechanism observed in females exposed to testosterone between gestational days 30 and 80 or between days 50 and 100 but not for shorter periods such as embryonic days 30–51 or 65–86 (25, 38).
The site and mechanism of estrogen’s influence remain uncertain. Sex differences do not exist in the number, distribution, or morphology of GnRH neurons in the rat and sheep (40, 41, 42). They have, however, been found in the synaptic innervation pattern of GnRH neurons (43), the immediate early gene transcriptional response of GnRH neurons to estradiol (44), and the expression of galanin by a subpopulation of GnRH neurons (45, 46). Although some of these observations implicate sex differences in the non-GnRH components of the network, their identity remains elusive (47, 48, 49).
B. Biosynthesis of GnRH
Available evidence suggests that the transcription rate, mRNA
stability, and posttranslational processing of GnRH are all actively
regulated in native GnRH neurons (50). With respect to the effects of
estrogen on GnRH biosynthesis, most attention has focused upon GnRH
mRNA expression, and several investigators have examined the influence
of the estrous cycle, ovariectomy, and estrogen replacement on
transcript levels in the female rat (reviewed in Refs. 50 and 51).
Unfortunately, the result of these studies has been one of widespread
disagreement as to whether estrogen has any influence on GnRH mRNA
expression and if so, whether this may be inhibitory or stimulatory in
nature (see tables in Refs. 50 and 51). However, recent work from the
laboratory of Petersen and co-workers (52, 53) has cleared much of this
confusion by showing that estrogen’s influence on GnRH mRNA expression
is dependent upon the time of day as well as the precise location of
the GnRH neurons within the medial septal-preoptic-hypothalamic
continuum. In essence, it appears that estrogen exerts a stimulatory
influence on GnRH mRNA expression in only rostral preoptic area neurons
and that a gradual increase in GnRH mRNA content, from the low levels
found in the morning in estrogen-treated rats, results in a peak of
expression in the early afternoon before the onset of the LH surge
(Fig. 1
). As GnRH mRNA expression in
ovariectomized rats remains relatively stable, at an intermediate level
between that of morning and afternoon levels in estradiol-treated rats
(Fig. 1), the evaluation of estrogen-treated rats killed in the morning
(54, 55), when estrogen is exerting an inhibitory influence on LH
secretion, seems likely to have been responsible for the reported
inhibitory effects of estrogen on GnRH mRNA content (53). Thus,
estrogen at low levels appears to inhibit GnRH mRNA expression relative
to ovariectomized rats while estrogen at higher concentrations and in
association with a circadian mechanism enhances GnRH mRNA levels (Fig. 1).
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Information on the estrogen regulation of GnRH mRNA in species other than the rat is very limited. Recent studies in the sheep suggest that cellular GnRH mRNA content does not increase in association with the GnRH surge (67, 68). This may represent an important species difference and, perhaps, reflect the absence of any substantial circadian influence upon the timing of the GnRH surge in this species. In terms of estrogen’s inhibitory influence upon GnRH transcript levels, a recent study suggests that estrogen may also repress GnRH mRNA expression in the human as postmenopausal females were found to exhibit significantly higher levels of cellular GnRH mRNA compared with premenopausal women, and this was argued to be independent of age (69). Our own preliminary work in the mouse also indicates that higher levels of GnRH mRNA expression are found throughout the GnRH population after gonadectomy (70).
Estrogen’s stimulatory influence upon GnRH mRNA expression is likely to be exerted principally at the level of GnRH gene transcription. Gore and Roberts (63) reported that an increase in GnRH primary transcript expression occurred early on proestrus, and Petersen and colleagues (64) found that estrogen increased GnRH primary transcript levels in advance of the increase in mature GnRH mRNA before the LH surge. It is less clear whether the inhibitory influence of estrogen on GnRH mRNA content may also be exerted at a transcriptional level. Two recent studies that have used GnRH promotor-driven reporters to examine the in vivo transcriptional regulation of the GnRH gene in transgenic mice have found that gonadectomy increases, while estradiol given in a negative feedback manner reduces, reporter expression in the rostral preoptic area (70, 71). While such evidence suggests that estrogen may alter gene transcription to both increase and decrease GnRH mRNA expression, the degree to which changes in GnRH mRNA stability (50) may be involved has not been established.
It is true, however, that the overall level of GnRH mRNA content
correlates with mean LH secretion better than it does with circulating
estradiol concentrations (Fig. 1
). This suggests that a simple linear
relationship between circulating estradiol concentrations and GnRH mRNA
expression is unlikely to exist and that multiple estrogen-receptive
and/or other pathways are likely to be involved in regulating GnRH gene
transcription and mRNA processing within the cell. For example, as
outlined above, there is good evidence in the rat for an interaction
between estrogen and circadian inputs on GnRH mRNA expression.
Studies also indicate that estrogen is involved in regulating the posttranslational processing of the pro-GnRH peptide. Ovariectomy has been found to decrease pro-GnRH peptide concentrations either with (57) or without (72) similar changes in GnRH mRNA expression, and estradiol replacement does not alter GnRH mRNA and pro-GnRH peptide levels to the same degree (57). Hence, changes in GnRH mRNA and pro-GnRH peptide contents do not necessarily occur in parallel, and this has led to the idea that estrogen may also regulate the posttranslational synthesis and/or processing of the pro-GnRH precursor (51, 57, 73).
C. Biosynthesis of galanin
The only well characterized neuropeptide known to be coexpressed
by GnRH neurons is the 29-amino acid neuropeptide galanin (74, 75).
Galanin mRNA and peptide are expressed in a sexually dimorphic manner
within a subpopulation of GnRH neurons (46, 76), and this depends upon
both organizational actions of testosterone during the perinatal period
(45, 46) and activational effects of estradiol in the adult female rat
(77, 78, 79, 80, 81). Work in the Steiner laboratory has shown that adult
ovariectomy results in a decrease in galanin mRNA expression by GnRH
neurons, and that this can be restored to intact levels by estrogen
replacement (77). Also, the galanin mRNA content of GnRH neurons is
maximal on the afternoon of proestrus (77) and, again, this has been
shown to result principally from a stimulatory action of circulating
estradiol (81). Although it is notable that workers in that laboratory
have never found concomitant changes in GnRH mRNA expression, their
reports of changing profiles of galanin mRNA content within GnRH
neurons have striking similarities with the changes in cellular GnRH
mRNA expression found by others (above). In particular, it seems that
estrogen may exert a stimulatory influence on the GnRH cell content of
both galanin and GnRH transcripts at the time of the estrogen-induced
LH surge. One difference is that the galanin mRNA content of GnRH
neurons remains elevated for more than 24 h (82) while GnRH
transcript numbers, possibly under the influence of progesterone, fall
shortly after the surge (53, 63). Whether this stimulatory action of
estrogen results from the transcriptional regulation of the galanin
gene, as it does for GnRH (63, 64), is not known. Although a good
correlation has been shown repeatedly between GnRH "activation" and
galanin mRNA expression within GnRH neurons, the physiological
significance of galanin’s presence within these cells remains unknown
(83).
D. Electrical activity of GnRH neurons
The present inability to make direct electrical recordings from
GnRH neurons in any routine manner has resulted in an almost complete
absence of any information about the effects of estrogen on GnRH
neuronal firing. The single exception has been work from the Kelly
laboratory (84, 85) where the immunocytochemical post-identification of
recorded neurons in the guinea-pig brain slice preparation has resulted
in the demonstration of rapid hyperpolarizing actions of 100
nM concentrations of estradiol on the membrane excitability
of small numbers of GnRH neurons. As these effects were observed in the
presence of tetrodotoxin, which blocks sodium channels and thus
synaptic connectivity, estrogen is likely to be acting directly upon
the GnRH neuron to reduce its level of electrical activity.
The "electrical" activity of GnRH neurons has also been assessed indirectly by examining expression patterns of immediate early genes such as c-fos and c-jun. Although not expressed by GnRH neurons under all circumstances of increased GnRH secretion, the presence of Fos protein within GnRH neurons is, in general, indicative of neuronal activation (86). Studies in the rat (86), mouse (87), and sheep (88), but curiously not the primate (89), have shown that many of the GnRH cells located within the rostral preoptic area and anterior hypothalamus express Fos at the time of the LH surge. In terms of understanding the estrogen inputs to the GnRH neurons, these observations are useful in that, like the GnRH mRNA studies, they define the rostral preoptic GnRH neurons of the rat as being particularly important in the generation of the GnRH/LH surge. Furthermore, independent studies in the rat have shown that, at the time of the surge, Fos is preferentially induced in GnRH neurons that receive likely circadian inputs (90) and express a relatively high GnRH mRNA content (91). Although estradiol replacement alone enables Fos to be expressed by GnRH neurons coincident with the LH surge, it is apparent that progesterone also plays a role as it enables the further recruitment of GnRH neurons to express Fos as the LH surge progresses in the rat (86). Together, the results of the Fos-GnRH studies are consistent with a stimulatory action of estrogen upon the GnRH cell bodies and suggest that estrogen and circadian inputs may converge to elevate cellular GnRH mRNA content in neurons of the rostral preoptic area in the rat. Although it is tempting to speculate that some of estrogen’s effects on GnRH or galanin gene transcription within the GnRH neuron are transduced by immediate early genes, their role(s) within these cells are unknown.
Another means of indirect assessment of GnRH neuron electrical activity has been the use of multiunit recording techniques to examine synchronized electrical discharges in the mediobasal hypothalamus (92, 93). Although these studies have been very successful in providing a good correlation between episodic bursts of multiunit activity and pulsatile LH secretion in a variety of species, the unknown origin of the electrical activity has made it difficult to interpret the absence of an increase in multiunit activity at the time of the LH surge (94, 95) and the estrogen-dependent changes in the size, shape, and occurrence of the multiunit volleys (96, 97, 98). An unanswered question of substantial importance to these studies is whether the multiunit activity results from action potentials in a subset, all, or none of the GnRH axons. There is now good evidence for GnRH receptor expression in the mediobasal hypothalamus of the rat (99), and GnRH is an extremely potent neurotransmitter in this area (100). Further, the infusion of GnRH into the mediobasal hypothalamus of the rat evokes multiunit activity (101). Hence, it is possible that multiunit activity reflects the postsynaptic activation of mediobasal hypothalamic neurons by GnRH collateral innervation. If so, this would represent one explanation for the excellent correlation of multiunit activity and pulsatile LH secretion in the ovariectomized animal, while the estrogen-induced changes in multiunit activity might better reflect the known gonadal steroid-dependent alterations in GnRH receptor expression by mediobasal hypothalamic neurons (99).
E. GnRH secretion
1. Stimulatory influence of estrogen
The use of push-pull sampling and portal bleeding techniques has
been extremely valuable in enabling investigators to describe the
effects of estrogen on GnRH secretion into the hypophyseal portal
system. There is wide agreement across species that estrogen exerts a
powerful stimulatory influence on GnRH secreted from nerve terminals in
the median eminence to initiate the LH surge (5, 6, 7, 8, 9, 15, 102, 103, 104). To
date, the best characterization of this estrogen-enhanced GnRH
secretion exists in the ewe (10) where the sampling of portal blood on
a minute-by-minute basis has been undertaken. Using this system, Evans
and colleagues (104, 105) have demonstrated that the first change from
the strictly episodic pattern of GnRH secretion observed under
estrogen’s negative feedback influence begins 15–20 h after estradiol
administration when significant nonepisodic interpulse GnRH secretion
occurs. This is followed by an increase in GnRH pulse frequency and
amplitude and further enhanced interpulse secretion in the 2- to 4-h
period before the onset of the ascending phase of the GnRH surge.
Hence, these investigators suggest that the stimulatory effects of
estrogen occur through a change in the nature of episodic GnRH
secretion as well as a gradual increase in nonepisodic GnRH release and
that, together, this represents a shift in the basic mode of GnRH
neurosecretion (105). Even in this species, however, there remains some
controversy as to whether the GnRH surge itself is composed of discrete
episodes of GnRH release or an outpouring of nonpulsatile GnRH (10, 103).
Although not analyzed in nearly the same detail, the description of the estrogen-induced GnRH surge in other species shows common basic features. In particular, it is important to note that the GnRH network of rat, sheep, and primate must be exposed to relatively high concentrations of estrogen for a prolonged period of around 15 h to enable a GnRH surge to occur (8, 11, 15). With respect to this 15-h period, an important recent insight (106) has been that termination of a 14-h estrogen signal before the onset of the LH surge has no effect on the duration or amplitude of the GnRH surge. This confirms further that prolonged estrogen exposure is required to initiate neural events that generate the GnRH surge but also emphasizes that estrogen need not be present at the time of the surge onset for it to occur. In contrast to the sheep and monkey, where estrogen is sufficient on its own to generate a normal GnRH and LH surge, the pituitary gonadotrophs and GnRH network of the rat require the presence of progesterone for the magnitude of the LH surge to resemble that of the proestrous event in the rat (see Ref. 19). Although the effects of progesterone supplementation on the estrogen-induced GnRH surge have not been established in the rat, indirect evidence such as the progesterone-dependent buildup of GnRH peptide in the median eminence on proestrus, and progesterone’s ability to advance the onset of the surge, suggests an effect of the steroid on GnRH secretion (14, 19). As estrogen is required to induce the expression of progesterone receptors, it is possible that estrogen and progesterone may exert synergistic actions upon the same components of the GnRH network immediately before and during the GnRH surge of the rat (20). After the surge, however, progesterone exerts an inhibitory influence on GnRH secretion in the rat (19), as found in other species.
2. Inhibitory influence of estrogen
Although much progress has been made in terms of describing the
stimulatory effect of estrogen on GnRH release, less agreement has
resulted from studies examining the inhibitory influence of estrogen.
Initial studies in the female monkey reported inconsistent elevations
in portal GnRH release after ovariectomy (107, 108) and failed to
detect any substantial effect of high concentrations of estradiol on
GnRH release in the median eminence/mediobasal hypothalamus (107, 109).
More recently, however, a relatively fast (2–3 h) and potent
inhibitory action of estrogen on GnRH pulse amplitude was reported in
the midpubertal monkey (12). Indirect assays of GnRH release in the
monkey have also suggested that part of the inhibitory effect of
estrogen on LH arises from an hypothalamic site (110). A similar state
exists in the rat, where Levine and Ramirez (111) found no effect of
ovariectomy on mediobasal hypothalamic GnRH release whereas Sarkar and
Fink (11), who were able to sample portal GnRH secretion, showed that a
significant increase in GnRH output occurred in long-term
ovariectomized rats and that estradiol rapidly reduced GnRH secretion
within 1 h. While detailed investigations of pulsatile GnRH
secretion have not been possible in the intact rat, it is worth noting
a series of studies by Leipheimer and colleagues (112, 113) in which
estradiol was found to be responsible for restraining both LH pulse
frequency and amplitude in intact diestrous rats.
More uniform results have been obtained in sheep where estradiol has been shown to exhibit clear inhibitory actions on portal GnRH secretion (7, 8, 13, 104). As also observed in the monkey (12), the inhibitory action of estrogen in the reproductively active sheep results principally from a decrease in GnRH pulse amplitude (13, 104). Few studies have undertaken a detailed time course analysis of how quickly estrogen suppresses GnRH pulse amplitude in the sheep, but published GnRH profiles suggest that it can occur as soon as 2 h after estrogen (7). Although the time interval after gonadectomy may be an important factor (11), it is unclear why the effects of gonadectomy and estrogen replacement have not been consistent within species such as the rat across different laboratories. It may also be relevant to note that the replacement of estradiol to physiological levels was found to suppress pulsatile GnRH secretion in an ovariectomized monkey while supraphysiological bolus injections of estradiol exerted no clear inhibitory action on GnRH (15).
F. GnRH degradation
Although not strictly an effect of estrogen on the GnRH neuron, it
is pertinent to note the emerging evidence that estrogen may also
regulate the degradation of GnRH upon release from the nerve terminal
in the median eminence. Several studies in the rat have reported that
the GnRH degradation rate fluctuates over the estrous cycle of the rat
and that peptidase activity is likely to be reduced at the time of the
proestrous or ovariectomized, steroid-induced LH surge (114, 115, 116, 117).
Others have shown that estradiol administration to ovariectomized rats
can increase (115, 118) or decrease (117) GnRH degradation rates in the
hypothalamus and/or pituitary gland.
More recent studies have isolated the individual peptidases acting on GnRH at the level of the median eminence and, while a number exist, the most important appears to be the zinc metalloendopeptidase-24.15 (EP 24.15; 119–121). Investigations by Wu and colleagues (122) have demonstrated the presence of EP 24.15 immunoreactivity associated with GnRH terminals in the median eminence and indicated that immunostaining intensity is reduced at the time of peak LH secretion during the surge. Although these workers were unable to show any fluctuations in portal blood levels of EP 24.15 on proestrus, the intravenous administration of a specific antagonist was found to increase the amplitude of the LH surge. In contrast, recent studies have failed to find a physiological role for several of the putative GnRH-active endopeptidases, including EP 24.15, in modulating the shape of GnRH pulses in the ovariectomized sheep (123). In that species, EP 24.15 activity in the median eminence was not found to alter across the estrous cycle although activity was reduced at the time of the estrogen-induced LH surge (123). These observations suggest that endopeptidases exert a physiological role in shaping the GnRH signal directed at the gonadotrophs at the time of the LH surge and that this may be regulated by gonadal steroids including estrogen.
G. Ultrastructural changes involving GnRH neurons
The GnRH neurons in the rat, sheep, and monkey exhibit a variety
of morphological types, are relatively sparsely innervated, and are
enveloped by glial cell processes to varying degrees (124, 125, 126, 127). There
is a substantial body of evidence that indicates that gonadal steroids
exert potent modulatory actions on the innervation patterns and glial
cell relationships of hypothalamic neurons (128), and a number of
studies have examined the GnRH neurons in this light. In the monkey,
Witkin and colleagues (126, 129) have shown that ovariectomy results in
a decrease in the percentage of GnRH neurons exhibiting a spiny
appearance, an increase in their degree of glial cell envelopment, and
a small decrease in their synaptic innervation. Preliminary data from
Naftolin and colleagues (130) using a different monkey model indicate,
however, that the chronic treatment of monkeys with premarin, an equine
estrogen preparation, results in a decrease in the number of synapses
on GnRH neurons.
In the rat, where there is less evidence for glial cell wrapping of the GnRH cell bodies, the ovariectomy of reproductively active females was found to have no effect on GnRH glial cell ensheathment or their density of synaptic input (131). At the level of the median eminence, ovariectomy was found to induce fluctuations in the distance between the GnRH terminals and the basal lamina overlying the portal capillaries in the rat (132). Furthermore, the GnRH terminals of the mouse and rat are known to be enveloped by tanycytic end feet (133, 134), and recent work by King and colleagues has suggested that gonadal steroids may modulate the degree of glial cell encasement of the GnRH terminals (134, 135). Hence, ultrastructural rearrangements in glial cell-GnRH terminal and cell body relationships clearly exist after gonadectomy. However, the degree to which they depend upon estrogen itself remains to be established.
H. Summary of estrogen’s influence on GnRH cells
Estrogen first impacts upon the GnRH neurons during development
when, after aromatization of testosterone, it acts to permanently
masculinize species-specific parameters of the GnRH network. In this
way, the "organizational" influence of estrogen engenders the
response of GnRH neurons to estrogen throughout postnatal life and
dictates that the masculinized network will respond differently to the
inhibitory influence of estrogen and/or be unable to generate a GnRH
surge.
In the adult female, estrogen exerts a stimulatory influence on the biosynthetic and secretory activity of the GnRH neurons. Estrogen regulates GnRH biosynthesis through modifications of gene transcription as well as posttranscriptional processing, and its stimulatory actions on GnRH secretion may involve the generation of a nonepisodic mode of GnRH release. The GnRH surge requires several hours of estrogen preexposure of the GnRH network and, in the rat, the presence of a circadian input. The GnRH neurons located in the rostral preoptic area of the rat appear to be particularly important in terms of estrogen’s stimulatory actions. The prolonged period of elevated estrogen exposure before ovulation seems likely to initiate a cascade of neural events that may differentially impact upon GnRH biosynthesis and secretion. These stimulatory effects require the presence of electrical activity in the brain as immediate early gene, galanin, and GnRH mRNA expression in GnRH neurons, as well as GnRH secretion itself, are all blocked by administration of the anesthetic pentobarbital (59, 78, 86).
The inhibitory effects of estrogen on GnRH neurons are more controversial, but evidence is consolidating in several species for an inhibitory influence on GnRH secretion, in particular GnRH pulse amplitude, as well as mRNA expression. Compared with estrogen’s stimulatory effects, this inhibitory influence on secretion can occur relatively quickly within 1–2 h and may correlate with the evidence for a direct inhibition of GnRH neuron electrical activity by estrogen in the guinea-pig.
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III. Brain Sites of Estrogen Influence on GnRH Neurons |
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TopAbstractI. IntroductionII. Effects of Estrogen...III. Brain Sites of...IV. Pathways for Estrogen...V. Model of Estrogen...VI. Concluding RemarksReferences |
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A. Sites of estrogen’s stimulatory action
1. The anteroventral periventricular nucleus (AVPv) in the
rat. The great majority of lesioning and estradiol implant studies
addressing the sites of action of estrogen in regulating LH secretion
have been undertaken in the rat. In terms of the LH surge, there is
wide agreement that the AVPv is the critical brain region within which
estrogen acts to enable the GnRH/LH surge to occur. This nucleus lies
within the rostral aspect of the medial preoptic area adjacent to the
third ventricle and immediately caudal to the organum vasculosum of the
lamina terminalis. It contains few, if any, GnRH neurons (139). Some
confusion has arisen in the literature over the naming of this nucleus
as it has been referred to as the anterior or rostral medial preoptic
nucleus as well as the medial preoptic nucleus, but is now widely
accepted as the AVPv (139, 140).
Lesions of the AVPv, but not neighboring nuclei, result in persistent estrus and the abolition of the estrogen- or estrogen plus progesterone-induced LH surge (141, 142, 143). Although not administered specifically into the AVPv, 17ß-estradiol placed in the medial preoptic area is able to generate an LH surge in the rat (144, 145) while similarly positioned implants of an antiestrogen inhibit the estrogen-induced LH surge, as well as the concomitant changes in GnRH mRNA expression (146, 147). The implantation of 17ß-estradiol or estradiol benzoate into the medial basal hypothalamus, amygdala, or hippocampus was not found to elicit a surge-like alteration in LH secretion (144, 145). These measurements in the rat involved LH rather than GnRH, and stimulatory actions of estradiol are well established to occur at the level of the gonadotroph (3, 4, 14). However, when estradiol concentrations reaching the pituitary gland are carefully controlled (145), the cerebral implantation data clearly supported the concept that the preoptic area was an important brain site through which estradiol likely initiated a GnRH surge in the rat (148).
Not surprisingly, receptor autoradiographic (149), immunocytochemical
(150, 151) and mRNA (152) analyses have all demonstrated a very
substantial population of ER-containing cells in the preoptic area
including the AVPv. The great majority of this work has concentrated on
the expression of ER
but, after the discovery of a second ER in
1996, termed ERß (153, 154), early studies are also suggesting the
existence of ERß mRNA (155), and to a lesser extent ERß protein
(156) in the AVPv. Anterograde tracing work shows that a population of
AVPv neurons project directly to GnRH neurons (157), and retrograde
tracing studies indicate that ER-expressing neurons of the AVPv
project to the immediate vicinity of the rostral preoptic GnRH neurons
(A. E. Herbison and S. X. Simonian, unpublished data). Together,
these observations indicate that estrogen-receptive AVPv neurons
project directly to the GnRH neurons and raise the possibility that
they may mediate the stimulatory actions of estrogen on the electrical
and biosynthetic behavior of the GnRH neurons. As ER-containing
neurons of the AVPv receive direct inputs from the suprachiasmatic
nucleus (158), they may also represent a site of integration of
circadian and estrogen inputs in the regulation of GnRH neurons.
The neurochemical identity of the estrogen-receptive AVPv neurons has
been examined in double-labeling studies, and subpopulations have been
reported to express a wide variety of neuropeptides in addition to the
inhibitory amino acid 
-aminobutyric acid (GABA) (Table 1). Interestingly, the AVPv is a sexually
dimorphic brain region exhibiting more cells in the female than male
(159, 160), and this is reflected in the numbers of ER-expressing
cells found in this area (151, 161, 162). Furthermore, some of the
neuropeptide-expressing neuronal populations in the AVPv are sexually
dimorphic with greater numbers of ER-containing calcitonin-gene
related peptide (163, 164), dynorphin (165, 166), neurotensin (NT)
(151), and substance P (162) neurons reported in the female compared
with the male. However, precisely which ER-containing neurons in the
AVPv project to the GnRH neurons remains unknown.
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-expressing cells of the
ventromedial nucleus generate a normal GnRH surge. Estradiol implants
within the ER-containing cells of the medial preoptic area were
unable to elicit a GnRH surge (167). This unexpected finding is in good
agreement, however, with previous work in which estradiol implants in
the vicinity of the ventromedial nucleus were found to evoke an LH
surge in the ewe (168). These observations are surprising as they
suggest that a substantial species difference may exist in the brain
sites used by estrogen to stimulate GnRH secretion. Earlier studies had
suggested that the rat and sheep might not have been different in this
respect as many neurons within the periventricular preoptic area of the
ewe, which may be the ovine AVPv equivalent, express Fos at the time of
the GnRH surge, and some of these neurons contained ERs (88). It will
be critical to establish the connectivity of the estrogen-receptive
neurons of the ovine ventromedial nucleus and also determine the brain
region(s) within the monkey involved in the estrogen-induced GnRH
surge.
B. Sites of estrogen’s inhibitory action
The brain sites involved in the inhibitory effects of estrogen on
GnRH neurons have been difficult to establish. Early studies in the rat
(169, 170) and monkey (171) documented inhibitory effects of estrogen
within the mediobasal hypothalamus on LH secretion, while others
working with the rat suggested that estradiol implants in the AVPv and
diagonal band of Broca inhibited LH pulse frequency in the
ovariectomized rat (172). A more recent study has shown that estrogen
implanted in the arcuate nucleus decreased LH pulse amplitude in
ovariectomized rats (173). However, the interpretation of the
mediobasal hypothalamus experiments may be confounded by actions of
estrogen diffusing to reach the pituitary and, in some cases, the
inaccuracies of single point LH sampling techniques (3, 145, 148).
Equally, however, the results with estradiol placements in the AVPv are
not consistent with the lesion studies and preoptic area antiestrogen
infusions experiments (142, 143, 146, 147), which abolished the LH
surge but failed to block estrogen’s inhibitory influence on LH
secretion. Recent studies in the sheep (167) have demonstrated that
both sites may have a role in the ovine species as 17ß-estradiol
implants located in either the medial preoptic area or arcuate nucleus
were found to inhibit the amplitude and frequency of pulsatile LH
secretion. A further complication in establishing the precise sites of
estrogen’s inhibitory influence comes from the ability of some brain
regions to exhibit an environment-dependent involvement in this
process. For example, estrogen administered into the paraventricular
nucleus will inhibit LH release in the fasting rat but not in the
normal or ovariectomized animal (174). Hence, existing data remain
inconclusive and suggest that multiple brain sites and mechanisms may
be involved, including the arcuate nucleus and preoptic area. Although
this state of progress may be revealing in its own right, this issue is
far from resolved.
Much less has been done in establishing the brain regions used by estrogen to inhibit GnRH mRNA levels. In hypothalamic slice cultures, estrogen was found to only inhibit GnRH mRNA expression when GnRH neurons existed in close association with the rostral preoptic area (175). In agreement with this observation, Petersen and colleagues demonstrated that the implantation of antiestrogens into the preoptic area blocked estrogen’s in vivo inhibitory influence on cellular GnRH mRNA content (147). Hence, the site of estrogen’s inhibitory influence upon GnRH biosynthesis may exist within the rostral preoptic area and may be the AVPv itself.
C. Summary
Nothing is known about the sites or mechanisms through which
estrogen sexually differentiates the GnRH network during development.
In the adult female rat, there is wide agreement that the AVPv with its
many estrogen-receptive neuronal cell populations (Table 1) is the
critical site at which estrogen acts to bring about its stimulatory
effects on GnRH release and biosynthesis. Neurons in the AVPv provide a
locus through which estrogen and circadian inputs can be integrated
before being transmitted to the GnRH neurons. The brain regions used by
estrogen to activate GnRH neurons in other species are less established
but may involve the mediobasal hypothalamus, and clear species
differences are evident.
The brain sites through which estrogen inhibits GnRH secretion are controversial but appear to be different to those used to stimulate GnRH neurons. More than one brain region, including the preoptic area and arcuate nucleus, may be involved. In contrast, available data indicate that the inhibitory effects of estrogen on GnRH mRNA expression may involve the preoptic area in the rat.
An important observation in the mouse (176, 177) and sheep (167) has been that the stimulatory and inhibitory influences of estrogen may occur independently of one another. For example, estradiol implants in the vicinity of the ovine ventromedial nucleus were found to evoke the GnRH/LH surge without showing any evidence of estradiol-negative feedback (167). Combined with evidence for different rates of onset of estrogen’s inhibitory and stimulatory actions on GnRH secretion, this suggests that different brain mechanisms may underlie these two phenomenon. Hence, it may be unhelpful to assume, as has previously been the case, that a "switch" from inhibitory to stimulatory feedback occurs. A more useful concept may be to suppose that two potentially independent estrogen-regulated mechanisms exist for restraining and activating GnRH activity (see Section V).
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IV. Pathways for Estrogen Regulation of GnRH Neurons |
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TopAbstractI. IntroductionII. Effects of Estrogen...III. Brain Sites of...IV. Pathways for Estrogen...V. Model of Estrogen...VI. Concluding RemarksReferences |
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). The first is that of a direct action of
estrogen on GnRH neuron electrical activity, the second an influence of
estrogen on other neurons within the GnRH network to regulate GnRH
neurons in a transsynaptic fashion, and the third involves changes in
glial-GnRH interactions. The following discussion examines each
mechanism in terms of its potential contribution to the inhibitory and
stimulatory influence of estrogen on GnRH secretion and biosynthesis.
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double-labeling immunocytochemistry in the rat
(151) and extended to the guinea pig (179), mouse (R. S. Slater and
A. E. Herbison, unpublished data), sheep (180, 181) and monkey
(182, 183). Although the lack of ER immunoreactivity alone is not
definitive evidence for the absence of ER in GnRH neurons, when
combined with the inability of GnRH neurons to concentrate estradiol,
this suggested strongly that estrogen was unlikely to regulate the
genome of the GnRH neuron directly in any mammalian species.
The very recent discovery of a second ER, termed ERß (153, 154), has
reopened the issue as to whether or not GnRH neurons may express an ER.
A recent study examining ERß mRNA expression in the rat brain (155)
has shown that it has a distribution in the rostral preoptic area
similar to that of ER mRNA (152). However, an initial
immunocytochemical study of ERß protein distribution in the rat brain
found relatively little staining in the preoptic area (156).
Furthermore, as the binding affinity of estradiol to ERß is very
similar to that of ER (184), the original observation by Shivers and
colleagues (178) that GnRH neurons do not concentrate
[3H]estradiol suggests that they may not express ERß
either. If so, it would appear that the presence of estrogen response
elements within the promotor region of the primate GnRH gene (185) are
not relevant in terms of GnRH expression in the brain and that effects
of estrogen on GnRH gene transcription do not occur through a classic
ER-mediated process. It is relevant to note, however, that a small
population of not more than 5% of GnRH neurons in the guinea pig have
been shown to express progesterone receptors (186).
The one caveat to these studies and the idea that ERs do not exist in GnRH neurons is that all of the investigations examining this issue have been performed in pubertal (183) or adult animals (151, 179, 180, 181, 182). As noted above, estrogen exerts dramatic and permanent effects on the GnRH network during development, and it remains possible that GnRH neurons express ERs during embryogenesis or early postnatal life and that ER synthesis is abruptly down-regulated with maturation. The one piece of evidence in support of this hypothesis is presence of ERs in immortalized GnRH-secreting cell lines (185, 187), which seem most likely to represent immature GnRH neurons (188, 189).
2. Nongenomic regulation. A substantial body of evidence
exists in support of rapid, nongenomic actions of estrogen upon neurons
located throughout the central nervous system (190, 191). The effects
and mechanisms of estrogen’s nongenomic actions appear diverse. Recent
work in the hippocampus indicates that nanomolar concentrations of
17ß-estradiol rapidly increase kainate receptor-induced currents by
altering the G protein-coupled, cAMP-dependent phosphorylation of
potassium channels (192). Similar concentrations of 17ß-estradiol
have also been shown to rapidly alter L-type calcium channels through a
putative G-protein coupled mechanism in the neostriatum (193). In both
the hippocampus and striatum, many of the effects of 17ß-estradiol
can be replicated with 17ß- estradiol conjugated to molecules such as
albumin and thus suggest that a membrane estradiol receptor may mediate
these effects. Within the hypothalamus, one of the best characterized
nongenomic influences of estradiol is that described by Lagrange and
colleagues (194, 195, 196) where nanomolar concentrations of estradiol
rapidly reduce the potency of both µ-opioid and GABAB
receptor agonists on mediobasal hypothalamic neurons. These
investigators propose that estrogen may bind to an ER-like receptor
to activate cAMP-dependent protein kinase which, in turn, uncouples
µ-opioid and GABAB receptors from inwardly rectifying
potassium channels (195, 196).
At present, the only similar data relating to the GnRH neurons is the demonstration of a direct hyperpolarizing influence of nanomolar 17ß-estradiol on GnRH neurons in the ovariectomized guinea pig (85). Unlike other mediobasal hypothalamic neurons in that species, acute estrogen treatment does not alter the response of GnRH neurons to µ-opioid receptor agonists (85), suggesting that GnRH neurons may not utilize the same estrogen-regulated G protein second messenger system. Clearly, much has yet to be done in determining the full extent of the nongenomic influences of estrogen on GnRH neurons and other neurons within the GnRH network. Estrogen is known to influence several of the enzyme components of the phosphoinositide and adenylase cyclase pathways (reviewed in Ref. 197), and this may underlie a wide range of possible nongenomic effects of estrogen within this network. It remains to be determined whether similar rapid effects of estrogen on GnRH neurons will occur in other species and whether physiological increments in estrogen concentrations, such as those seen before the LH surge, will evoke similar changes in GnRH membrane excitability. Furthermore, will the rapid, nongenomic effects of estrogen be confined to alterations in membrane physiology or also involve changes in gene transcription? In this light, it is of note that high concentrations of estrogen have been shown to increase phosphorylation of cAMP response element-binding proteins in the AVPv (198).
B. Estrogen-receptive neurons as intermediaries
Of all of the potential pathways through which estrogen may
regulate the activity of GnRH neurons, that involving ER-containing
neurons as intermediaries has received most attention. This has been
due, in part, to the difficulty of examining the other possibilities,
but also comes from the substantial amount of work indicating that
estrogen can act at brain sites remote from the GnRH neurons to
influence their activity (Section III). The identification
of multiple neurochemically distinct nerve terminals synapsing on GnRH
cell bodies (127) combined with the limited evidence for direct
presynaptic inputs to GnRH terminals in the median eminence (199, 200)
suggests that neurons may act directly at both sites to modulate GnRH
secretion. As estrogen-receptive neurons have been identified to
project to the immediate vicinity of the GnRH cell bodies in both rat
(201) and sheep (A. E. Herbison and A. Caraty, unpublished) as
well as the median eminence of the sheep (202) and monkey (203), it is
possible that ER-expressing neuronal populations influence GnRH neurons
at both terminal and cell body levels. A very extensive literature
exists with regard to the neurochemical regulation of LH and GnRH
secretion (204). However, strong data exist for only a few
neurotransmitter pathways in the transsynaptic mediation of estrogen’s
influence on GnRH neurons. It is clear that an understanding of the
estrogen-sensitive AVPv neurons would help greatly in elucidating the
nature of estrogen’s stimulatory actions on GnRH neurons of the rat.
Of the neurotransmitter-containing cell populations identified to
express ERs in this area (Table 1), NT and GABA are presently the most
likely candidates for a role in providing estrogen inputs to the GnRH
neurons. Data implicating dopaminergic neurons are weak in the rat but
strong in the anestrous ewe. Although the estrogen regulation of
enkephalin and dynorphin neurons in the AVPv is established, their
relationship to the estrogen regulation of GnRH activity is not yet
determined (166). Outside of the AVPv there has been a longstanding
interest in the role of the brainstem norepinephrine (NE) neurons (see
Ref. 201) and substantial investigation into the neuropeptide Y and
ß-endorphin (ßEND) populations of the arcuate nucleus (see Ref.
19). Finally, although the location of their cell bodies is unknown,
glutamate and aspartate cannot be ignored on the basis that they are
the most important excitatory neurotransmitters in the brain. A
synopsis for each of these neurochemicals in the estrogen-dependent
regulation of the GnRH network in adult females is presented below.
1. Dopamine. There is little agreement over the role, if any,
of dopamine in regulating LH secretion or GnRH activity in the rat
(204). As such, it is impossible to determine with any certainty the
role of dopamine in mediating estrogen influences within the GnRH
network of this species. This is despite the suggestion that
dopaminergic neurons of the AVPv project to GnRH neurons (205),
although these cells do not appear to express ER immunoreactivity in
the rat (Table 1).
This situation is in stark contrast to the sheep where substantial data agree that dopamine plays an important role in mediating estrogen’s inhibitory influence on GnRH secretion during anestrus (4, 206, 207). In the sheep, seasonal changes evoke a marked change in the sensitivity of the GnRH network to estradiol, with maximal estrogen-dependent inhibition exhibited in anestrus (208). Dopamine D2 receptors are clearly involved in the estrogen-dependent suppression of LH pulse frequency in anestrous ewes (209, 210, 211, 212), and lesion studies suggest that this involves the A14 and/or A15 dopaminergic cell populations of the anterior hypothalamus (213, 214). Furthermore, recent investigations have shown that both A14 and A15 cell populations are activated by estrogen during anestrus but not during the breeding season (215, 216). Although it is known that the season-dependent inhibition of GnRH secretion by dopamine occurs at the level of the median eminence (199, 217, 218), the relationship of the A14 and A15 dopamine neurons to the relevant dopaminergic terminals in the median eminence remains obscure (216, 219).
The mechanisms through which estrogen influences the ovine A14/15
dopaminergic neurons to inhibit pulsatile GnRH secretion in a seasonal
manner are not yet established. Although ER immunoreactivity can be
demonstrated in the A14, but not A15, dopaminergic neurons, the number
of A14 cells expressing ER does not change across season (220).
Furthermore, in contrast to the seasonal changes in tyrosine
hydroxylase enzyme activity within the median eminence (218), no
consistent differences in tyrosine hydroxylase mRNA, immunoreactivity,
or enzyme activity have been detected in the A14 or A15 cell bodies
(218, 220, 221). The absence of ERs in the A15 dopaminergic neurons and
lack of changes in cell body tyrosine hydroxylase activity suggest that
estrogen may influence the electrical activity of these cells either
through their afferent inputs or in a nongenomic manner. Thus, although
it is not known exactly how estradiol influences these dopaminergic
neurons to regulate GnRH secretion in anestrous sheep, they remain an
interesting illustration of how the GnRH network can recruit specific
estrogen-receptive neural components in an environmentally dependent
manner.
2. Excitatory amino acids. The amino acids glutamate and
aspartate are important excitatory neurotransmitters within the
neuroendocrine hypothalamus (222) and exert powerful actions on the
output of the GnRH network (reviewed in Ref. 223). Studies employing
the pharmacological blockade of the N-methyl-D-aspartate
(NMDA), DL--amino-3-hydroxy-5-methyl-4-isoxazole
propionic acid (AMPA), and kainate receptors have shown that glutamate
and aspartate play a role in generating both the pulsatile (224) and
surge (225, 226, 227) modes of GnRH/LH secretion in the adult female rat.
Hence, it seems highly likely that neurons utilizing excitatory amino
acids are part of the GnRH network.
However, one of the major problems in trying to understand the excitatory amino acid component of the GnRH network is our present inability to identify the location of the glutamatergic cell bodies. As all cells use glutamate for biosynthetic purposes, it is difficult to determine which neurons employ it specifically as a neurotransmitter. This problem is complicated further by the presence of NMDA, AMPA, and kainate receptor subtypes in all of the brain regions implicated in the network (reviewed in Ref. 223). Only small subpopulations of GnRH neurons in the rat have been reported to express NMDA and/or kainate receptors (228, 229), and terminals containing glutamate have been demonstrated to synapse on GnRH neurons in the monkey (230). Neurons throughout the preoptic area and hypothalamus, including the AVPv, have been shown to express all three types of ionotropic excitatory amino acid receptors (223, 231, 232). Hence, while the phenomenon of excitatory amino acid involvement within the GnRH network is clear, the mechanism of glutamate and aspartate action within the network is likely to be difficult to establish.
These restrictions make it difficult to decipher whether or not excitatory amino acids are involved in mediating the inhibitory and/or stimulatory actions of estrogen within the GnRH network. It is quite clear, for example, that the facilitatory effect of NMDA, and AMPA (223), on LH secretion is dependent upon the presence of estrogen in the adult rat, mouse, sheep, and monkey and that it does not involve the pituitary gland (233, 234, 235, 236). Hence the NMDA and AMPA receptors and/or their related second messenger pathways appear to be "enabled" by estrogen acting in a negative feedback manner on LH secretion. Where these receptors are found, and whether they are actually involved in mediating the inhibitory influence of estrogen within the GnRH network, are unknown. Excitatory amino acid release in the preoptic area and mediobasal hypothalamus is not different in ovariectomized rats and those given estrogen to inhibit LH secretion (237, 238, 239). Adding further to this dilemma was the observation that NMDA was equally potent in stimulating GnRH release from mediobasal hypothalamic explants in ovariectomized oil- and estrogen-replaced rats (235). As studies have not been conducted to determine whether the blockade of excitatory amino acid receptor activity influences estrogen’s inhibitory actions on LH, it is not possible, as yet, to comment on their involvement in this mechanism.
There is better evidence to implicate excitatory amino acids in the relay of estrogen’s stimulatory influence on GnRH secretion at the time of the surge. The LH surge in rats is clearly blocked or attenuated by excitatory amino acid receptor antagonists, and this is not a pituitary effect (225, 226, 227). Furthermore, glutamate and aspartate release within the vicinity of the GnRH cell bodies, but not the GnRH terminals, is seen to increase at the time of the surge (237, 238, 239). There is presently disagreement, however, as to the relative roles of estrogen and progesterone in the generation of this increased release (237, 238, 239). Nevertheless, it appears likely that the GnRH neurons, or cells in the vicinity of the preoptic area, are targets for increased excitatory amino acid release at the time of the surge and that estrogen is involved. By analogy with the magnocellular oxytocin network (240), excitatory amino acids may provide the excitatory signal responsible for initiating the GnRH surge.
The excitatory amino acids may also be involved in stimulating GnRH mRNA expression at the time of the LH surge (241). However, previous studies (reviewed in Ref. 50) have shown that NMDA receptor activation enhances GnRH mRNA stability rather than altering GnRH gene transcription, which is the principal mode of estrogen’s stimulatory effect on cellular GnRH mRNA levels at this time (Section II.B).
3. -Aminobutyric acid. There is substantial evidence for a
role of the amino acid GABA within the GnRH network. Terminals
synapsing on GnRH neurons contain GABA (242), GnRH neurons express
GABAA receptors (243), and in vivo microinfusion
data indicate that GABA inputs within the vicinity of the GnRH cell
bodies exert a powerful inhibitory influence on the secretory activity
of GnRH neurons in several species (244, 245, 246, 247, 248). The location of ER in
preoptic GABA neurons (180, 249, 250) and the influence of estradiol on
preoptic GABA release (251, 252, 253), re-uptake (254), and
GABAA receptor expression (255) suggests that the preoptic
GABA network of the rat is highly sensitive to circulating estrogens
(250). Recent evidence also indicates a high level of ER expression in
GABA neurons found within the vicinity of GnRH neurons in the pubertal
monkey (256).
It is possible, therefore, that estrogen-sensitive GABA neurons have a role within the GnRH network in mediating part of the inhibitory effects of estrogen on GnRH neurons. Indeed, an inverse relationship exists between preoptic GABA levels and mean LH release in the rat; preoptic GABA release is low in ovariectomized rats that exhibit high LH output (251) and increased when estrogen exerts its inhibitory action on LH secretion (252, 253). A similar inverse profile of GABA levels and GnRH secretion has been detected in the prepubertal and pubertal monkey (248). Importantly, GABAA receptor blockade has been shown to elevate LH release in estrogen-treated rats (257) and anestrous sheep (247) exhibiting negative feedback. Together, these observations suggest that estrogen-receptive GABA neurons within the preoptic area, including the AVPv of the rat, may be partly responsible for mediating estrogen’s inhibitory effects on GnRH secretion.
Another important observation has been that GABA concentrations within the vicinity of the GnRH cell bodies, but not GnRH terminals, fall before the onset of the LH/GnRH surge in the rat and sheep (238, 258, 259). This is thought to be a prerequisite for the occurrence of the LH surge in the rat (246, 260). However, the relationship and mechanism of estrogen’s influence, if any, on GABA neurons before the LH surge is less clear. Although estrogen does not influence the activity or mRNA expression of glutamic acid decarboxylase (the GABA synthetic enzyme) in the medial preoptic area of the rat (reviewed in Ref. 250), the decrease in GABA levels before the LH surge is known to correlate with a decrease in glutamic acid decarboxylase mRNA expression in neurons of this area (261, 262). Recent studies suggest that this fall in glutamic acid decarboxylase mRNA expression results from the rise in progesterone secretion before the start of the LH surge (263). Equally, however, the fall in GABA concentrations within the vicinity of the GnRH cell bodies may result from circadian inputs within the GnRH network in the rat. One further influence of estrogen within the preoptic GABA population is its ability to uncouple the normal stimulatory influence of NE on GABA neurons, and thereby allow NE release to increase while allowing that of GABA to fall at the time of the surge (264). Hence, there is clear evidence of an important dis-inhibition of GnRH neurons by GABA at the time of the surge (246, 260, 265), but this looks to be a multifactorial response to estrogen, progesterone, and, possibly, circadian influences in the rat.
The actions of GABA on GnRH neurons may not be restricted solely to the regulation of GnRH secretion, as recent studies have documented effects of pharmacological GABA receptor activation and blockade on GnRH mRNA expression in the female rat (266, 267, 268). The reported effects have not, however, been consistent. In the ovariectomized rat, GABAA receptor activation was found to have either no effect (266) or to decrease (268) GnRH mRNA content while GABAB receptor activation was also shown to have no effect (268) or to decrease (266) GnRH transcript levels. Hence, at this stage, it is not possible to determine whether the inhibitory effect of estrogen on GnRH mRNA content, suggested to be mediated by the AVPv (Section III.B), involves preoptic GABA projections to the GnRH neurons.
4. Neuropeptide Y (NPY). NPY acts at the level of the GnRH
cell bodies and terminals as well as at the gonadotrophs of the
pituitary gland and has a clear role in facilitating both GnRH release
and gonadotroph responsiveness to GnRH at the time of the LH surge (19, 20). The NPY innervation of the hypothalamus and preoptic area
originates from essentially two cell populations, one located in the
arcuate nucleus and the other in the brainstem (269, 270). In terms of
mediating estrogen’s influence within the GnRH network, it is the NPY
neurons located in the arcuate nucleus that are most likely to be
regulated in a classic genomic manner by estrogen. Approximately 10%
of NPY neurons in the arcuate nucleus express ERs in the rat (19) and
sheep (220), and NPY mRNA expression in this region is increased by
estradiol (271) and elevated during proestrus (272). In contrast, the
NPY-containing neurons of the brainstem have not been found to express
ER immunoreactivity (273).
Most evidence indicates that the median eminence is the principal site at which estrogen induces the stimulatory effect of NPY on GnRH neurons. In association with estrogen’s enhancement of NPY mRNA expression in the arcuate nucleus (271), an increase in NPY content (19) and release (274) is observed in the median eminence before and during the GnRH/LH surge of the rat. Further, NPY stimulates GnRH release from the median eminence, and this response has been shown to be potentiated by estrogen (275) and maximal at proestrus in rats (276). A similar role for NPY in influencing GnRH release has been established in the primate (277, 278) and may also exist in the ewe (279). In the rat, the NPY responses of GnRH terminals and gonadotrophs appear to be initiated by estrogen and then enhanced further by progesterone secreted in advance of the LH surge (19, 280, 281). Hence, it seems not unreasonable to suggest that part of the facilitatory influence of estrogen on GnRH neurons at the time of the surge occurs through an elevation of NPY synthesis and secretion in the median eminence in addition to a likely sensitization of GnRH terminals to the stimulatory action of NPY.
The mechanisms underlying the effects of estrogen on the NPY receptors are unclear as ligand binding to the NPY Y1 receptor in the hypothalamus, which mediates the effects of NPY on GnRH release (19, 20), was shown recently to be unaffected by estrogen or progesterone administration (282). Furthermore, it is not known whether GnRH neurons express the Y1 receptor. Although a direct effect of estrogen on the ER-expressing NPY cells of the arcuate nucleus seems likely to be involved in elevating NPY release in the median eminence, estrogen may also act indirectly by reducing opioid inhibition of these cells before the surge (283).
Actions of NPY on the GnRH network outside the median eminence are also
likely to exist but their physiological relevance is less clearly
understood. Nerve terminals containing NPY have been found to synapse
on GnRH cell bodies in the rat (284) and sheep (285), but not the
monkey (286), and NPY concentrations within the vicinity of the GnRH
cell bodies increase on proestrus (287) and during the
estradiol-induced LH surge in the rat (288). Recent observations using
retrograde tracing techniques indicate that NPY inputs to the immediate
vicinity of the GnRH cell bodies in the rostral preoptic area come from
neurons located in both the brainstem and arcuate nucleus and that a
small number of the arcuate NPY neurons contain ER (A. E.
Herbison and S. X. Simonian, unpublished data). However, the effects of
this potential estrogen-dependent increase in NPY input to the GnRH
cell bodies are unknown. Data in the male rat suggest a role for NPY in
regulating GnRH gene expression (289).
Overall, however, the prime importance of the AVPv in mediating estrogen’s stimulatory input within the GnRH network of the rat (Section III.A.1) should be borne in mind. The placement of estrogen implants in the arcuate nucleus is insufficient to generate an LH surge whereas implants in the preoptic area alone create a normal estrogen-induced surge (144, 145). Hence, in this light, the NPY neurons of the arcuate nucleus would seem very unlikely to provide all of the stimulatory gonadal steroid input required by the GnRH neurons to induce a surge. The AVPv is known to project massively to the arcuate nucleus (157), and it is possible that the AVPv coordinates the estrogen signal within the GnRH network by recruiting different components of the network such as, in this case, the arcuate NPY neurons to modulate secretion from the GnRH terminals and pituitary gonadotrophs.
5. Neurotensin. A role for NT as a stimulatory input to the GnRH network has evolved through a series of studies that have shown that NT acts within the vicinity of the GnRH cell bodies to regulate the magnitude of the LH surge. The infusion of NT into the preoptic area increases the height of the LH surge (290, 291) while the administration of NT antisera reduces the size of the LH surge (292). As neither treatment influences the timing of the LH surge, and there is no evidence for NT to modulate gonadotroph functioning (293), NT neurons represent a stimulatory component of the GnRH network that may be independent of the circadian trigger in the rat.
Although the preoptic area has been defined as the site of NT’s
stimulatory influence within the GnRH network of the rat (291, 292, 294), it remains unclear whether this involves the GnRH neurons
directly (293). The location of the NT-synthesizing cell bodies that
project to the preoptic area are not known. However, the foregoing
discussion regarding the role of the AVPv in transmitting estrogen
input to the GnRH neurons (Section III.A.1) makes the
observation of a large population of NT neurons within the AVPv (295, 296) relevant. This is especially so as ER immunoreactivity has been
detected in AVPv NT neurons (151, 297), and estrogen is known to
increase AVPv NT peptide and mRNA expression (292, 296). Hence, one
plausible scenario would involve the elevated estrogen concentrations
near the time of the surge stimulating AVPv NT neurons to release
greater amounts of NT on GnRH neurons. However, nothing is known at
present about changes in NT concentrations in the vicinity of the GnRH
cell bodies. A study showing that the stimulatory effect of NT on LH
could be blocked by the prior administration of catecholaminergic
blockers (298) indicates that the NE component of the GnRH network
needs to be functional for NT to exert its stimulatory action. It does
not demonstrate that the effects of NT are mediated by catecholamines.
NT cells are found in other regions of the hypothalamus (299), and
those located in the arcuate nucleus also express ER
immunoreactivity (D. P. Spratt and A. E. Herbison, unpublished
data) and respond to estrogen by increasing their NT mRNA content
(300). Many of these neurons coexpress dopamine and project to the
median eminence (301, 302) from where NT release has been shown to be
regulated by estrogen (303). A role has been proposed for
estrogen-dependent NT release from the median eminence in regulating
PRL secretion while its involvement within the GnRH network is unknown
(293).
6. Norepinephrine. NE is likely to exert a permissive
influence favoring high output states of the GnRH neuron within the
GnRH network (Ref. 201 for recent review) and several studies have
suggested that the brainstem NE neurons play a role in mediating part
of estrogen’s stimulatory actions within the GnRH network. A good
correlation exists between estradiol status and NE turnover or release
in the preoptic area and median eminence (304, 305, 306, 307, 308, 309, 310), and NE
concentrations in these areas are elevated before, or in association
with, the occurrence of the LH surge (304, 307, 308, 309, 310). Further, the
acute effect of noradrenergic pathway ablation (311, 312, 313) or
administration of -adrenergic receptor antagonists is to block the
occurrence of the LH surge (314, 315, 316).
An important observation in the NE ablation studies has been that the LH surge mechanism can exhibit full recovery several weeks after the lesioning (311, 312). As suggested by a number of investigators and argued again recently (201), this is good evidence for the permissive nature of NE within the GnRH network. It should not be regarded as a conventional inhibitory or excitatory drive, but rather as a neuromodulator that enables interactions within the network depending upon its local concentrations. Hence, as a part of the GnRH network that is not involved directly in determining the mode of GnRH output (201), NE’s permissive actions should be theoretically replaceable under pathological conditions such as brain lesioning when a degree of neuronal plasticity may be evident. Equally, however, by acting as a permissive global influence under normal physiological conditions, the NE component of the network would represent a particularly effective manner through which estrogen could facilitate the output of the GnRH neurons.
Recent investigations have begun to establish the precise NE pathways
through which estrogen influences the GnRH network. A number of
experimental approaches in the rat have provided evidence of
projections from NE neurons to the preoptic area (317, 318, 319, 320, 321), and, more
recently, Wright and Jennes (322) demonstrated that NE neurons in both
the ventrolateral medulla (A1 neurons) and nucleus tractus solitarii
(A2 neurons) project to the vicinity of the GnRH cell bodies.
Subpopulations of these brainstem A1 and A2 neurons express ER in
the rat (273, 323), and triple-labeling studies have now gone on to
show that ERs are located in A2, but not A1, neurons which project to
the immediate vicinity of rostral preoptic GnRH neurons in the rat
(201). Although the pattern of ER expression in A1 and A2 neurons of
the sheep is very similar to that of the rat (324), the
estrogen-receptive NE inputs in this species may be different as there
is relatively little evidence for A2 projections to the preoptic area
in the ovine brain (325).
Although the exact relationship of A1 and A2 inputs to GnRH neurons
remains uncertain, they are likely to involve both direct and indirect
actions. Electron microscopic investigations have found nerve terminals
containing tyrosine hydroxylase synapsing on GnRH cell bodies (326, 327), but no studies have demonstrated positively that these inputs
contain NE rather than dopamine (127). Nevertheless, a recent
investigation has reported the presence of 2A-adrenergic
receptor immunoreactivity in native GnRH neurons (328), and this
indicates that NE neurons may form direct synapses with GnRH cells. It
should be noted, however, that the stimulatory influence of NE within
the GnRH network is mediated by 1-adrenergic receptors
(61, 315, 316, 329). Evidence for the indirect regulation of GnRH
neurons by NE also exists and may involve, at least, a serial
arrangement between NE, GABA, and GnRH neurons in the rat (264, 326, 330). The location and exact targets of NE neurons that may influence
GnRH secretion at the level of the nerve terminals within the median
eminence are unknown (331).
The precise manner in which estrogen influences NE neurons is not yet established. Estrogen has been demonstrated to increase the electrical excitability of NE neurons projecting to the preoptic area (332) and also to stimulate Fos (333) and tyrosine hydroxylase (334) gene expression in A2 neurons. Furthermore, the number of A2 neurons expressing Fos increases on proestrus before the onset of the LH surge (333, 335). Hence, estrogen may facilitate a diurnal pattern of NE secretion within the preoptic area by stimulating the A2 subpopulation to elevate NE release in the morning/early afternoon of proestrus (307, 335). In addition, there is good evidence for a presynaptic inhibition of NE release by ßEND in the preoptic area of the rat, and an estrogen-induced decrease in this tonic input may further contribute to the elevation of preoptic NE concentrations at the time of the LH surge (336, 337, 338, 339). Together, these data indicate that the A2 neurons projecting to the immediate vicinity of the GnRH cell bodies are likely to be stimulated directly by estrogen and that, in association with a reduced presynaptic opioid restraint, are responsible for the increased NE release within the rostral preoptic area. Much less is known about the gonadal steroid-induced changes in NE release within the median eminence, although it is notable that turnover in this region seems to be particularly sensitive to changes in serum progesterone concentrations (305).
Estrogen also alters adrenergic receptor expression and functioning
within the preoptic area and hypothalamus. It has been shown to exert a
relatively selective stimulatory influence upon the 1B
adrenergic receptor subtype (340), and studies have reported that
estrogen enhances 1b adrenoceptor mRNA (341) to increase
the density of these receptors within the preoptic area and
hypothalamus. This is thought to be involved in the estrogenic
modulation of second messenger pathways in these cells (197, 342).
Also, the electrical responses of neurons located in the arcuate
nucleus to -adrenergic agonists are enhanced by estrogen treatment
(343). How these receptor changes relate to alterations in the activity
of the GnRH network is not clear. There is long-standing evidence for
NE exerting inhibitory effects on LH secretion in ovariectomized
animals and stimulatory actions on LH release in estrogen-treated rats
(344, 345, 346). However, this exact "inhibition-stimulation" phenomenon
has now been observed in response to many different neurotransmitters
and neuropeptides (19, 223). As such, it is possible that the
phenomenon does not result from any unique attribute of the individual
neurochemical but, rather, represents gonadal steroid-dependent
differences in the underlying properties and behavior of the GnRH
neurons themselves and/or their most important primary afferents.
The estrogen-dependent increase in NE release upon cells of the preoptic area and median eminence in proestrous rats is likely to alter both GnRH secretion and biosynthesis. Although much evidence indicates that NE promotes GnRH secretion, the cellular mechanisms through which this may occur are not well established. Work in the male rat has suggested that NE stimulates GnRH secretion within the median eminence by inducing the synthesis of nitric oxide and prostaglandin E2 (347, 348, 349). Elsewhere in the brain, studies suggest that NE may facilitate electrical activity within a network by increasing the signal-to-noise ratio of specific synapses and even act as a ‘gate’ to enable silent excitatory inputs to function (350). Such electrophysiological actions would be in good agreement with the proposed permissive influence of NE within the GnRH network where it may function to "enable" neurotransmission between components.
In terms of the potential for NE to influence GnRH mRNA expression, it
is worth noting that the increase in NE concentrations within the
vicinity of the GnRH cell bodies occurs before the onset of the LH
surge (307, 308, 310). Importantly, the estrogen-induced increase in
GnRH mRNA content in rostral preoptic neurons is blocked by
pretreatment with an 1-adrenergic receptor antagonist
(61). As cellular GnRH mRNA content can be elevated as little as 1
h after intraventricular NE administration (351), the endogenous NE
increase in the preoptic area on proestrus appears well positioned in a
temporal sense to aid in the increased expression of GnRH mRNA, which
occurs before the onset of the LH surge (53, 62). However, it is
unclear at present whether this rapid NE-induced elevation in cellular
GnRH mRNA content results from an increase in GnRH gene transcription,
which is the principal mode of estrogen action upon GnRH mRNA content
at this time (63, 64).
7. Opioid peptides. Although substantial populations of
enkephalin- and dynorphin-containing cell populations exist within the
mediobasal hypothalamus and preoptic area, and some of these are
regulated by gonadal steroids (166, 352, 353), there is little work
indicating whether they may be involved in transmitting estrogen input
within the GnRH network. In the ovariectomized rat, dynorphins were
shown to suppress LH release by acting through 
-opiate receptors
while met-enkephalin exerted the same action via 
-receptors (354, 355). Light microscope immunocytochemical studies have shown
enkephalin- and dynorphin-containing fibers in the vicinity of the GnRH
cell bodies and terminals in the female rat (356). However, studies
examining the involvement of - and/or -opiate receptors in
mediating estrogen’s inhibitory and stimulatory effects on LH
secretion have not been undertaken (19).
In contrast, a substantial literature has built up around the role of
ßEND and µ-opiate receptors within the GnRH network and their role
in transmitting estrogen input. Neurons synthesizing ßEND are found
exclusively within the region of the arcuate nucleus, and a small
percentage (5–10%) have been shown to concentrate estradiol (357, 358) and express immunoreactive ER in the rodent (D. P. Spratt and
A. E. Herbison, unpublished) and sheep (181). The likelihood of
direct transsynaptic regulation of the GnRH neurons by ßEND is
supported by the demonstration of ßEND-containing terminals synapsing
on the dendrites and soma of GnRH neurons in the female rat (43, 359)
and monkey (360) and direct actions of a µ-opiate receptor agonist on
the electrical activity of GnRH neurons in the guinea pig (85). These
observations are thought to be functionally relevant as infusions of
µ-opiate receptor agonists or antagonists into the preoptic area
alter LH release in vivo (361, 362, 363). However, the recent
finding that µ-, -, and -opiate receptor mRNAs are all
detectable within rostral preoptic area cells but not GnRH neurons,
when assessed using dual labeling in situ hybridization
(364), has questioned whether ßEND may, indeed, be acting directly
upon GnRH neurons. Certainly, evidence exists for both NE (19, 338) and
excitatory amino acids (365) as mediators of opioid peptide influences
on LH secretion. Further, some pharmacological data suggest that opioid
peptides may act within the mediobasal hypothalamus to influence GnRH
secretion (361, 366). Hence, the ßEND neurons are most likely to
exert both direct and indirect actions on GnRH neurons within the
network.
Brain lesion and implant studies suggest that neurons located in the arcuate nucleus may represent one pathway through which the inhibitory influence of estrogen is exerted on GnRH secretion (Section III.B). The potent inhibitory actions of ßEND and µ-opiate receptor agonists upon LH release in ovariectomized rats and the ability of naloxone, an opiate receptor antagonist, to elevate the low level of LH secretion observed in the presence of estrogen (354, 355, 362, 367, 368) suggested that the arcuate ßEND neurons may subserve just such a role. However, subsequent studies revealed that naloxone stimulated GnRH and LH secretion in a manner that was independent of gonadal steroid status in rats as well as ewes (369, 370, 371). Hence, despite early promise, the ßEND neurons have now been shown not to mediate estrogen’s inhibitory influence on GnRH secretion. Rather, the naloxone data indicate that the ßEND neurons form a component of the GnRH network that maintains a continuous inhibitory tone within the network and that this may be involved in determining the dynamics of individual GnRH pulses (371).
There is, nevertheless, an important role proposed for ßEND in mediating some of the stimulatory effects of estrogen on GnRH neurons in rats, sheep, and primates (19, 372). Consistent results from several laboratories have shown that a substantial reduction in opioid activity occurs within the GnRH network preceding the LH surge. Whereas naloxone administration reveals a tonic inhibitory opioid tone at other times of the ovarian cycle, it fails to alter LH secretion when administered in the afternoon before the natural or estrogen-induced surge commences (373, 374, 375). This, along with data showing a reduction in ßEND peptide content in the preoptic area and mediobasal hypothalamus of rats and sheep (372) and a fall in ßEND release from the median eminence on proestrus (376), indicates that a reduction in ßEND release occurs within the GnRH network before the LH surge occurs. Changes are also evident in µ-opiate receptor function as, unlike other times of the cycle, morphine exerts no effects upon LH secretion when administered before the surge (368). Furthermore, a fall in naloxone binding occurs on the afternoon of the LH surge in the preoptic area and median eminence (377, 378). Together these observations indicate that a relatively brief period of reduced ßEND release and µ-opiate receptor activity occurs at the time of the LH surge to dis-inhibit the GnRH network.
Estrogen has an important role in bringing about this presurge dis-inhibition by decreasing both opioid receptor binding (379) and ßEND release (372, 380, 381, 382, 383, 384). In terms of the latter, it is clear that prolonged estrogen exposure decreases ßEND mRNA and peptide expression within the arcuate nucleus (380, 382, 383). However, ßEND peptide and mRNA analyses also show that a significant diurnal rhythm occurs within the ßEND neurons; ßEND expression is lowest in the afternoon, and this diurnal rhythm is only evident in the presence of estrogen (381, 384, 385). Hence, the arcuate ßEND neurons may be an additional site at which estrogen and circadian inputs are integrated before transmission to the GnRH neurons in the rat. Such an hypothesis is in agreement with experiments that have shown that the artificial reduction of opiate activity on the afternoon of proestrus can advance the onset of the LH surge (386, 387, 388). Thus, it seems that one of the mechanisms underlying the stimulatory influence of estrogen upon GnRH secretion is to enable ßEND neurons to exhibit a diurnal decline in their level of biosynthetic and secretory activity and, like the preoptic GABA neurons, dis-inhibit the GnRH neurons at the time of the surge.
As with a number of other estrogen-induced processes at the time of the LH surge in the rat (19), progesterone facilitates the effects of estrogen and initially promotes the afternoon decline in ßEND activity within the GnRH network (19, 386, 388, 389). However, the marked increase in progesterone concentrations that follows the onset of the LH surge exerts an opposite effect to estrogen on these cells and increases ßEND mRNA levels (381, 383). This is suggested to be the neural mechanism through which progesterone inhibits the occurrence of a further estrogen-induced LH surge on estrus (386, 389). Progesterone has similarly been suggested to utilize ßEND neurons to inhibit LH secretion in the sheep (4).
The neural pathways through which circadian and estrogen inputs
regulate the ßEND neurons to influence GnRH neurons in the rat are
not entirely clear. Neural inputs from the suprachiasmatic nucleus to
the arcuate are known in the rat (390, 391, 392) and may well underlie the
diurnal activity of these neurons. It is more difficult, however, to
envisage how estrogen exerts its genomic influence upon this cell
population as only approximately 5% of these cells concentrate
estradiol (357) or express immunocytochemically detectable ER (D. P.
Spratt and A. E. Herbison, unpublished), and ERß is not reported
to be in abundance within the arcuate nucleus of the rat (155, 156).
Hence, estrogen may regulate the gene expression and secretory activity
of ßEND neurons through mechanisms that are indirect. This issue is
emphasized further by our recent unpublished observation that none of
the arcuate ßEND neurons projecting to the immediate vicinity of
rostral preoptic area GnRH neurons express ER immunoreactivity. Yet
again, it is worth emphasizing that the arcuate nucleus is insufficient
on its own to mediate the stimulatory effects of estrogen within the
GnRH network (Section III.A). Hence, as suggested for NPY,
it may be that the estrogen-receptive neurons of the AVPv have a role
in transsynaptically influencing the activity of the arcuate ßEND
neurons. As highlighted above, the precise nature of ßEND
interactions with GnRH neurons is unclear.
C. Glial cells as intermediaries
There is growing support for an important structural and chemical
interaction between glial cells and GnRH neurons (393). Glial cells
surround GnRH cell bodies, and alterations in this ensheathment may
underlie the associated changes in synaptic input to GnRH neurons
reported after gonadal steroid manipulations (126). Equally, gonadal
steroid-dependent alterations in the level of encasement of GnRH
terminals by tanycytic end-feet could play an important role in
regulating the dynamics of GnRH release into the portal vasculature
(134). The lack of directly relevant data makes it difficult to ascribe
a role for these structural alterations in either the inhibitory or
stimulatory effects of estrogen within the GnRH network. However, it is
pertinent that estrogen increases the growth and branching of glial
cell processes (394, 395) and has been reported to exert relatively
fast (5 h), reversible effects on the glial cell ensheathment of
neurons in the arcuate nucleus (396). In this light, it would be very
useful to know whether estrogen exerts similar effects on the
GnRH-glial relationship.
The evidence for bidirectional influences of chemical messengers
between glial cells and GnRH neurons arises almost entirely from
in vitro studies using GnRH-secreting neuronal lines such as
the GT1 cells (393). Such studies have identified a range of epidermal
growth factor family members that are secreted by glial cells and found
to influence the differentiation and/or activity of GnRH neurons. For
example, basic fibroblast growth factor is thought to act directly upon
GT1 cells to enhance neuronal differentiation and GnRH processing
(397, 398, 399) while transforming growth factor- and -ß1 are involved
in regulating GnRH secretion in an indirect manner (399, 400, 401), possibly
through the production of prostaglandin E2 and other
molecules from glial cells (402). In terms of estrogen regulation, an
important observation made by Langub and Watson (403) was that
astrocytes and ependymal cells express ER in the guinea pig
hypothalamus. Hence, glial cells within these areas are likely to have
the ability to be influenced directly by estrogen, and recent data
indicate that estrogen acts on glial cells to cause the secretion of
unidentified factors which enhance prostaglandin E2
receptor subtype expression in GT1 neurons (404). Although it is
impossible, at present, to ascribe any role of estrogen-induced glial
cell secretions to the actions of estrogen on native adult GnRH
neurons, these observations in GT1 cells may be particularly relevant
to the sexually differentiating effects of estrogen during GnRH
development.
D. Summary
Estrogen uses a variety of different mechanisms to regulate the
biosynthetic and secretory activity of GnRH neurons (Table 2).
|
and Table 2). The excitatory amino
acids, NT and NE, are all used by estrogen to activate GnRH electrical
activity at the level of the GnRH cell body, while NPY and NE have a
similar role within the median eminence, presumably on GnRH terminals.
Estrogen also utilizes the ßEND neurons but, in this case, reduces
their level of activity to dis-inhibit GnRH neurons. It is less clear
whether the GABA-ergic dis-inhibition, also occurring at this time, is
driven primarily by estrogen. Hence, estrogen appears to orchestrate
the activity patterns of several neurotransmitter systems within the
GnRH network to bring about the GnRH surge. At present, there is little
primary evidence for estrogen to activate GnRH neurons directly or
alter glial cell interactions to elevate GnRH secretion (Table 2). The
one anomaly in this scenario is that the NT input is the only component
that could be regarded as originating from the AVPv, the principal site
at which estrogen is thought to act to initiate the GnRH surge in the
rat. It is suggested that the very substantial projections from the
AVPv to the arcuate nucleus may represent an important pathway through
which estrogen coordinates NPY and ßEND neurons to help induce the
surge. The NE component of the network should be regarded as a
permissive neuromodulatory element. Quite clearly, each of these
interconnected, yet parallel, components must be functional for the
GnRH surge to occur; the acute pharmacological disruption of any one
will prevent its occurrence. The prolonged period of estrogen exposure
required for the GnRH surge to occur is entirely consistent with the
generation by estrogen of a coordinated cascade of events mediated by
ER-dependent alterations in gene transcription. In the rat, there is
potential for the circadian and estrogen inputs to be integrated at the
level of the ER-expressing AVPv neurons and/or the ßEND cells of the
arcuate nucleus.
|
). As a neuronal
input originating from the preoptic area is implicated in increasing
GnRH mRNA expression in response to estrogen, the critical neural
population may not yet have been identified.
In terms of the inhibitory actions of estrogen on GnRH neurons, there
is less solid evidence for the involvement of estrogen-receptive
interneuron populations (Table 2). Of all the neural populations
reviewed, only GABA can be postulated to be involved in the inhibition
of GnRH secretion, and ßEND has been positively excluded. This is
despite the LH response to many different neurochemicals being
inhibitory in the absence of estrogen and stimulatory in its presence.
There is convincing evidence in the guinea pig that estrogen inhibits
GnRH secretion through a direct membrane action. This observation is
compatible with evidence that estradiol can inhibit LH secretion when
placed in the immediate vicinity of the GnRH neurons and that estrogen
exerts relatively rapid inhibitory effects on GnRH release. As altered
ultrastructural glial-GnRH relationships are observed when comparing
intact and gonadectomized animals, estrogen may also act at this level
to inhibit activity within the GnRH network. Hence, present data
indicate that estrogen may use multiple different modes of action to
repress GnRH secretion into the portal circulation (Table 2).
There is almost no information available on the mechanisms through
which estrogen may inhibit GnRH mRNA expression (Table 2).
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V. Model of Estrogen Action on GnRH Neurons |
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TopAbstractI. IntroductionII. Effects of Estrogen...III. Brain Sites of...IV. Pathways for Estrogen...V. Model of Estrogen...VI. Concluding RemarksReferences |
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). This is
suggested by evidence of 1) different brain sites involved in bringing
about the two modes of estrogen action, 2) different rates of
estrogen’s stimulatory and inhibitory influences, and 3) data
indicating that the inhibitory and stimulatory actions can occur
independently of one another (Section III.C).
|
and Table 2) and are envisaged to occur through an
initial rapid and direct hyperpolarization of GnRH neurons followed by
an increase in preoptic GABA release from estrogen-sensitive
interneurons within hours and then structural rearrangements in glial
cell interactions with GnRH cell bodies and terminals over hours to
days. As such, the inhibitory actions of estrogen would occur rapidly
but ultimately involve several anatomically distinct components with
differing temporal onset (and offset). It is of note that much
difficulty has been experienced in identifying a single brain region
mediating the inhibitory effects of estrogen on GnRH neurons
(Section III.B) and that a considerable degree of
fluctuation in GnRH output occurs with time after gonadectomy (see Ref.
51). Once established, the multimodal nature of the inhibitory input
would suggest a relatively stable form of feedback on a day-to-day
basis. Nevertheless, in situations such as the seasonally breeding ewe,
where the sensitivity of the GnRH neurons to estrogen’s inhibitory
actions is clearly enhanced during the months of anestrus, new neural
populations such as the A15 dopaminergic neurons may be recruited
specifically at this time to enhance negative feedback actions of
estrogen. Hence, the proposed estrogen-sensitive inhibitory pathway would account for the relatively fast actions of estrogen in reducing GnRH secretion, the difficulties faced in trying to establish a single brain region mediating these actions, and the varying influence of time after ovariectomy on GnRH output. The ability to add and remove additional components to the pathway would enable the longer term modulation of estrogen’s inhibitory influence within the network.
In contrast to the inhibitory pathway, the stimulatory effects of
estrogen are proposed to be unimodal and involve principally the
transsynaptic regulation of GnRH neurons by estrogen-sensitive neurons
(Fig. 4). At present there is good evidence for the involvement of
several neurotransmitter populations in bringing about the stimulatory
actions of estrogen (Fig. 3), but no data on rapid membrane actions and
little direct evidence for ultrastructural glial-GnRH changes (Table 2). Indeed, the recent evidence that elevated estrogen concentrations
need not be present at the time of surge onset itself (106) suggests
that direct rapid actions of estrogen on GnRH have no role at this
time. The prolonged 15-h period of estrogen exposure required to
generate the GnRH surge is entirely consistent with a classic genomic
action of estrogen to alter gene expression and drive a cascade of
temporally distinct but coordinated neurochemical activities at
different levels within the GnRH network to alter gene expression. The
increasing concentrations of estradiol before the surge would thus
enhance the synthesis and release of stimulatory neurotransmitters such
as the excitatory amino acids, NT, NPY, and NE, while decreasing the
synthesis and release of inhibitory neurotransmitters such as ßEND,
and possibly GABA. In rats, the circadian input within the GnRH network
is only seen to involve estrogen’s stimulatory aspects and would, as
evidence suggests (Section IV.B), interact with neuronal
populations implicated in the stimulatory pathway.
The main feature of this model is that it does not rely on a
"switch" from the so-called negative to positive feedback actions
of estrogen. Instead, it is suggested that the stimulatory
transsynaptic pathway is driven by the fluctuating estrogen
concentrations and that this predominates over a relatively constant
multimodal inhibitory pathway for a short period each cycle to evoke
the GnRH surge (Fig. 4). Hence, estrogen would use direct,
transsynaptic, and glial cells’ modes of action to establish a
relatively restrained pattern of pulsatile GnRH secretion, and possibly
GnRH biosynthesis, whereas the stimulatory effects of estrogen would
result from the cyclical activation of specific neuronal cell
populations, which would intermittently superimpose their influence
upon GnRH secretion and biosynthesis to generate the GnRH surge. Data
in both the rat (20) and sheep (10) would support the concept that a
surge-specific pattern of GnRH secretion superimposes itself upon the
normal pulsatile profile of GnRH release observed under estrogen’s
inhibitory influence. In the rat, estrogen-induced progesterone
receptors, transactivated in a ligand-dependent or -independent manner
(20), would appear to facilitate the effects of estrogen within the
ER-expressing neuronal components of the GnRH network at the beginning
of the surge. Thus, at any one time, it is proposed that the mode of
GnRH secretion will reflect the net balance of estrogen-regulated
inhibitory and stimulatory pathway influences within the GnRH network
(Fig. 4).
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VI. Concluding Remarks |
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TopAbstractI. IntroductionII. Effects of Estrogen...III. Brain Sites of...IV. Pathways for Estrogen...V. Model of Estrogen...VI. Concluding RemarksReferences |
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Acknowledgments |
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Footnotes |
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1 Lister Institute-Jenner Fellow. 
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References |
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TopAbstractI. IntroductionII. Effects of Estrogen...III. Brain Sites of...IV. Pathways for Estrogen...V. Model of Estrogen...VI. Concluding RemarksReferences |
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S. Kanda, Y. Akazome, T. Matsunaga, N. Yamamoto, S. Yamada, H. Tsukamura, K.-i. Maeda, and Y. Oka Identification of KiSS-1 Product Kisspeptin and Steroid-Sensitive Sexually Dimorphic Kisspeptin Neurons in Medaka (Oryzias latipes) Endocrinology, May 1, 2008; 149(5): 2467 - 2476. [Abstract] [Full Text] [PDF]
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W. Huang, M. Acosta-Martinez, and J. E. Levine Ovarian Steroids Stimulate Adenosine Triphosphate-Sensitive Potassium (KATP) Channel Subunit Gene Expression and Confer Responsiveness of the Gonadotropin-Releasing Hormone Pulse Generator to KATP Channel Modulation Endocrinology, May 1, 2008; 149(5): 2423 - 2432. [Abstract] [Full Text] [PDF]
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 Q. Gao and T. L. Horvath Cross-talk between estrogen and leptin signaling in the hypothalamus Am J Physiol Endocrinol Metab, May 1, 2008; 294(5): E817 - E826. [Abstract] [Full Text] [PDF]
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G. M. Anderson, D. C. Kieser, F. J. Steyn, and D. R. Grattan Hypothalamic Prolactin Receptor Messenger Ribonucleic Acid Levels, Prolactin Signaling, and Hyperprolactinemic Inhibition of Pulsatile Luteinizing Hormone Secretion Are Dependent on Estradiol Endocrinology, April 1, 2008; 149(4): 1562 - 1570. [Abstract] [Full Text] [PDF]
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 G. Rasier, A.-S. Parent, A. Gerard, R. Denooz, M.-C. Lebrethon, C. Charlier, and J.-P. Bourguignon Mechanisms of Interaction of Endocrine-Disrupting Chemicals with Glutamate-Evoked Secretion of Gonadotropin-Releasing Hormone Toxicol. Sci., March 1, 2008; 102(1): 33 - 41. [Abstract] [Full Text] [PDF]
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H. Abe, K. L. Keen, and E. Terasawa Rapid Action of Estrogens on Intracellular Calcium Oscillations in Primate Luteinizing Hormone-Releasing Hormone-1 Neurons Endocrinology, March 1, 2008; 149(3): 1155 - 1162. [Abstract] [Full Text] [PDF]
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J. Clasadonte, P. Poulain, J.-C. Beauvillain, and V. Prevot Activation of Neuronal Nitric Oxide Release Inhibits Spontaneous Firing in Adult Gonadotropin-Releasing Hormone Neurons: A Possible Local Synchronizing Signal Endocrinology, February 1, 2008; 149(2): 587 - 596. [Abstract] [Full Text] [PDF]
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G. Rasier, A.-S. Parent, A. Gerard, M.-C. Lebrethon, and J.-P. Bourguignon Early Maturation of Gonadotropin-Releasing Hormone Secretion and Sexual Precocity after Exposure of Infant Female Rats to Estradiol or Dichlorodiphenyltrichloroethane Biol Reprod, October 1, 2007; 77(4): 734 - 742. [Abstract] [Full Text] [PDF]
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C. Zhang, M. A. Bosch, J. E. Levine, O. K. Ronnekleiv, and M. J. Kelly Gonadotropin-Releasing Hormone Neurons Express KATP Channels That Are Regulated by Estrogen and Responsive to Glucose and Metabolic Inhibition J. Neurosci., September 19, 2007; 27(38): 10153 - 10164. [Abstract] [Full Text] [PDF]
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A. Morales, M. Gonzalez, R. Marin, M. Diaz, and R. Alonso Estrogen inhibition of norepinephrine responsiveness is initiated at the plasma membrane of GnRH-producing GT1-7 cells J. Endocrinol., July 1, 2007; 194(1): 193 - 200. [Abstract] [Full Text] [PDF]
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 C. J McManus, M. Valent, S. L Hardy, and R. L Goodman Does nitric oxide act in the ventromedial preoptic area to mediate oestrogen negative feedback in the seasonally anoestrous ewe? Reproduction, July 1, 2007; 134(1): 137 - 145. [Abstract] [Full Text] [PDF]
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X. d'Anglemont de Tassigny, C. Campagne, B. Dehouck, D. Leroy, G. R. Holstein, J.-C. Beauvillain, V. Buee-Scherrer, and V. Prevot Coupling of Neuronal Nitric Oxide Synthase to NMDA Receptors via Postsynaptic Density-95 Depends on Estrogen and Contributes to the Central Control of Adult Female Reproduction J. Neurosci., June 6, 2007; 27(23): 6103 - 6114. [Abstract] [Full Text] [PDF]
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C. Glidewell-Kenney, L. A. Hurley, L. Pfaff, J. Weiss, J. E. Levine, and J. L. Jameson Nonclassical estrogen receptor {alpha} signaling mediates negative feedback in the female mouse reproductive axis PNAS, May 8, 2007; 104(19): 8173 - 8177. [Abstract] [Full Text] [PDF]
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A. S. Kauffman, M. L. Gottsch, J. Roa, A. C. Byquist, A. Crown, D. K. Clifton, G. E. Hoffman, R. A. Steiner, and M. Tena-Sempere Sexual Differentiation of Kiss1 Gene Expression in the Brain of the Rat Endocrinology, April 1, 2007; 148(4): 1774 - 1783. [Abstract] [Full Text] [PDF]
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 A. R. Genazzani, N. Pluchino, S. Luisi, and M. Luisi Estrogen, cognition and female ageing Hum. Reprod. Update, March 1, 2007; 13(2): 175 - 187. [Abstract] [Full Text] [PDF]
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J. A. Taylor, M.-L. Goubillon, K. D. Broad, and J. E. Robinson Steroid Control of Gonadotropin-Releasing Hormone Secretion: Associated Changes in Pro-Opiomelanocortin and Preproenkephalin Messenger RNA Expression in the Ovine Hypothalamus Biol Reprod, March 1, 2007; 76(3): 524 - 531. [Abstract] [Full Text] [PDF]
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C. A. Christian and S. M. Moenter Estradiol Induces Diurnal Shifts in GABA Transmission to Gonadotropin-Releasing Hormone Neurons to Provide a Neural Signal for Ovulation J. Neurosci., February 21, 2007; 27(8): 1913 - 1921. [Abstract] [Full Text] [PDF]
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C A Wilson and D C Davies The control of sexual differentiation of the reproductive system and brain Reproduction, February 1, 2007; 133(2): 331 - 359. [Abstract] [Full Text] [PDF]
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B. Furnes and J. Schimenti Fast forward to new genes in mammalian reproduction J. Physiol., January 1, 2007; 578(1): 25 - 32. [Abstract] [Full Text] [PDF]
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J. Clarkson and A. E. Herbison Postnatal Development of Kisspeptin Neurons in Mouse Hypothalamus; Sexual Dimorphism and Projections to Gonadotropin-Releasing Hormone Neurons Endocrinology, December 1, 2006; 147(12): 5817 - 5825. [Abstract] [Full Text] [PDF]
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J. Qiu, M. A. Bosch, K. Jamali, C. Xue, M. J. Kelly, and O. K. Ronnekleiv Estrogen Upregulates T-Type Calcium Channels in the Hypothalamus and Pituitary J. Neurosci., October 25, 2006; 26(43): 11072 - 11082. [Abstract] [Full Text] [PDF]
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 V. Rochira, L. Zirilli, A. D Genazzani, A. Balestrieri, C. Aranda, B. Fabre, P. Antunez, C. Diazzi, C. Carani, and L. Maffei Hypothalamic-pituitary-gonadal axis in two men with aromatase deficiency: evidence that circulating estrogens are required at the hypothalamic level for the integrity of gonadotropin negative feedback. Eur. J. Endocrinol., October 1, 2006; 155(4): 513 - 522. [Abstract] [Full Text] [PDF]
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 D. Titolo, F. Cai, and D. D. Belsham Coordinate Regulation of Neuropeptide Y and Agouti-Related Peptide Gene Expression by Estrogen Depends on the Ratio of Estrogen Receptor (ER) {alpha} to ER{beta} in Clonal Hypothalamic Neurons Mol. Endocrinol., September 1, 2006; 20(9): 2080 - 2092. [Abstract] [Full Text] [PDF]
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J. T. Smith, S. M. Popa, D. K. Clifton, G. E. Hoffman, and R. A. Steiner Kiss1 neurons in the forebrain as central processors for generating the preovulatory luteinizing hormone surge. J. Neurosci., June 21, 2006; 26(25): 6687 - 6694. [Abstract] [Full Text] [PDF]
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M. El-Etr, Y. Akwa, E.-E. Baulieu, and M. Schumacher The Neuroactive Steroid Pregnenolone Sulfate Stimulates the Release of Gonadotropin-Releasing Hormone from GT1-7 Hypothalamic Neurons, through N-Methyl-D-Aspartate Receptors Endocrinology, June 1, 2006; 147(6): 2737 - 2743. [Abstract] [Full Text] [PDF]
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J. Pielecka and S. M. Moenter Effect of Steroid Milieu on Gonadotropin-Releasing Hormone-1 Neuron Firing Pattern and Luteinizing Hormone Levels in Male Mice Biol Reprod, May 1, 2006; 74(5): 931 - 937. [Abstract] [Full Text] [PDF]
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J. T Smith, D. K Clifton, and R. A Steiner Regulation of the neuroendocrine reproductive axis by kisspeptin-GPR54 signaling. Reproduction, April 1, 2006; 131(4): 623 - 630. [Abstract] [Full Text] [PDF]
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 J. D. Veldhuis, J. N. Roemmich, E. J. Richmond, and C. Y. Bowers Somatotropic and Gonadotropic Axes Linkages in Infancy, Childhood, and the Puberty-Adult Transition Endocr. Rev., April 1, 2006; 27(2): 101 - 140. [Abstract] [Full Text] [PDF]
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H. O. de la Iglesia and W. J. Schwartz Minireview: Timely Ovulation: Circadian Regulation of the Female Hypothalamo-Pituitary-Gonadal Axis Endocrinology, March 1, 2006; 147(3): 1148 - 1153. [Abstract] [Full Text] [PDF]
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L. J. Kriegsfeld, D. F. Mei, G. E. Bentley, T. Ubuka, A. O. Mason, K. Inoue, K. Ukena, K. Tsutsui, and R. Silver Identification and characterization of a gonadotropin-inhibitory system in the brains of mammals PNAS, February 14, 2006; 103(7): 2410 - 2415. [Abstract] [Full Text] [PDF]
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C. V. V. Helena, M. de Oliveira Poletini, G. L. Sanvitto, S. Hayashi, C. R. Franci, and J. A. Anselmo-Franci Changes in {alpha}-estradiol receptor and progesterone receptor expression in the locus coeruleus and preoptic area throughout the rat estrous cycle J. Endocrinol., February 1, 2006; 188(2): 155 - 165. [Abstract] [Full Text] [PDF]
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S. Pompolo, A. Pereira, K. M. Estrada, and I. J. Clarke Colocalization of Kisspeptin and Gonadotropin-Releasing Hormone in the Ovine Brain Endocrinology, February 1, 2006; 147(2): 804 - 810. [Abstract] [Full Text] [PDF]
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J. Balthazart, M. Baillien, and G. F. Ball Rapid Control of Brain Aromatase Activity by Glutamatergic Inputs Endocrinology, January 1, 2006; 147(1): 359 - 366. [Abstract] [Full Text] [PDF]
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C. Bodo, A. E. Kudwa, and E. F. Rissman Both Estrogen Receptor-{alpha} and -{beta} Are Required for Sexual Differentiation of the Anteroventral Periventricular Area in Mice Endocrinology, January 1, 2006; 147(1): 415 - 420. [Abstract] [Full Text] [PDF]
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S.-K. Han, M. L. Gottsch, K. J. Lee, S. M. Popa, J. T. Smith, S. K. Jakawich, D. K. Clifton, R. A. Steiner, and A. E. Herbison Activation of Gonadotropin-Releasing Hormone Neurons by Kisspeptin as a Neuroendocrine Switch for the Onset of Puberty J. Neurosci., December 7, 2005; 25(49): 11349 - 11356. [Abstract] [Full Text] [PDF]
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S. M. Moenter and R. A. DeFazio Endogenous {gamma}-Aminobutyric Acid Can Excite Gonadotropin-Releasing Hormone Neurons Endocrinology, December 1, 2005; 146(12): 5374 - 5379. [Abstract] [Full Text] [PDF]
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C. A. Christian, J. L. Mobley, and S. M. Moenter Diurnal and estradiol-dependent changes in gonadotropin-releasing hormone neuron firing activity PNAS, October 25, 2005; 102(43): 15682 - 15687. [Abstract] [Full Text] [PDF]
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J. T. Smith, M. J. Cunningham, E. F. Rissman, D. K Clifton, and R. A. Steiner Regulation of Kiss1 Gene Expression in the Brain of the Female Mouse Endocrinology, September 1, 2005; 146(9): 3686 - 3692. [Abstract] [Full Text] [PDF]
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J. T. Smith, H. M. Dungan, E. A. Stoll, M. L. Gottsch, R. E. Braun, S. M. Eacker, D. K Clifton, and R. A. Steiner Differential Regulation of KiSS-1 mRNA Expression by Sex Steroids in the Brain of the Male Mouse Endocrinology, July 1, 2005; 146(7): 2976 - 2984. [Abstract] [Full Text] [PDF]
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I. Merchenthaler, G. E. Hoffman, and M. V. Lane Estrogen and Estrogen Receptor-{beta} (ER{beta})-Selective Ligands Induce Galanin Expression within Gonadotropin Hormone-Releasing Hormone-Immunoreactive Neurons in the Female Rat Brain Endocrinology, June 1, 2005; 146(6): 2760 - 2765. [Abstract] [Full Text] [PDF]
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V. Matagne, M.-C. Lebrethon, A. Gerard, and J.-P. Bourguignon Kainate/Estrogen Receptor Involvement in Rapid Estradiol Effects in Vitro and Intracellular Signaling Pathways Endocrinology, May 1, 2005; 146(5): 2313 - 2323. [Abstract] [Full Text] [PDF]
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J. M Russell, E Murphree, J Janik, and P Callahan Effect of steroids and nitric oxide on pituitary hormone release in ovariectomized, peripubertal rats Reproduction, April 1, 2005; 129(4): 497 - 504. [Abstract] [Full Text] [PDF]
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 M.B Hawkins, J Godwin, D Crews, and P Thomas The distributions of the duplicate oestrogen receptors ER-{beta}a and ER-{beta}b in the forebrain of the Atlantic croaker (Micropogonias undulatus): evidence for subfunctionalization after gene duplication Proc R Soc B, March 22, 2005; 272(1563): 633 - 641. [Abstract] [Full Text] [PDF]
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R. E. Campbell, S.-K. Han, and A. E. Herbison Biocytin Filling of Adult Gonadotropin-Releasing Hormone Neurons in Situ Reveals Extensive, Spiny, Dendritic Processes Endocrinology, March 1, 2005; 146(3): 1163 - 1169. [Abstract] [Full Text] [PDF]
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C M Gomes, C Raineki, P R. de Paula, G S Severino, C V V Helena, J A Anselmo-Franci, C R Franci, G L Sanvitto, and A B Lucion Neonatal handling and reproductive function in female rats J. Endocrinol., February 1, 2005; 184(2): 435 - 445. [Abstract] [Full Text] [PDF]
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 B.-S. An, J.-H. Choi, K.-C. Choi, and P. C. K. Leung Differential Role of Progesterone Receptor Isoforms in the Transcriptional Regulation of Human Gonadotropin-Releasing Hormone I (GnRH I) Receptor, GnRH I, and GnRH II J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 1106 - 1113. [Abstract] [Full Text] [PDF]
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