Download New Concepts of the Neuroendocrine Regulation of Gonadotropin

BIOLOGY OF REPRODUCTION 56, 293-302 (1997)
New Concepts of the Neuroendocrine Regulation of Gonadotropin Surges in Rats
Jon E. Levine'
Department of Neurobiology & Physiology, Northwestern University, Evanston, Illinois 60208
ABSTRACT
In species that ovulate spontaneously, two key events mediate the stimulation of preovulatory gonadotropin surges: 1) neurosecretion of a preovulatory LHRH surge and 2) an acute increase in responsiveness of the pituitary gland to the LHRH neurosecretory trigger. These processes, in turn, depend upon both
the positive feedback actions of preovulatory estrogen secretions
and specific neural signals for initiation of the surge. In female
rats, the neural signals for the surge are principally derived from
the 24-h neural clock, thereby limiting the timing of surges to
the afternoon of proestrus. It remains unclear, however, how
neural signals converge with endocrine signals (estrogen) inspecific brain cells and how their cellular integration leads to appropriate secretion of gonadotropin surges. Previous work has
suggested that estrogen may exert its facilitatory actions by
opening a neural "gate," thereby allowing transmission of the
daily neural signal to surge-initiating neuronal groups. How may
estrogen act to render a neural pathway patent? A conventional
view holds that steroid hormones can exert permissive effects
on signaling efficacy by modulating neurotransmitter receptor
expression, intracellular second messenger production, and protein kinase activity. However, recent evidence has suggested that
estrogen may also have the capacity to permit cross-talk between neurotransmitter signaling pathways and parallel transcriptional regulatory pathways. The progesterone receptor isan
estrogen-inducible transcription factor that has been shown to
be transactivated-even in the absence of its cognate ligandafter stimulation of neurotransmitter receptors coupled to adenylate cyclase stimulation. Thus, the convergence of neural and
endocrine signals for the stimulation of gonadotropin surges
could occur at the level of the progesterone receptor: estrogen
may stimulate expression of progesterone receptors, which in
turn may be initially transactivated by synaptic signals. Activated
progesterone receptors may thereafter regulate transcription of
target genes that control transmitter synthesis and release in
neural circuitries governing LHRH gene expression and/or pulsatile LHRH release. An analogous mechanism may operate in
pituitary gonadotrophs, in which ligand-independent transactivation of progesterone receptors mediates integration of neurosecretory and estrogen positive feedback signals, leading to
increased pituitary responsiveness to LHRH. It is proposed that
the "seeding" of specific neuronal groups and pituitary gonadotrophs with progesterone receptors, and perhaps other inducible transcription factors, comprises an important basis of estrogen's permissive role in the stimulation of gonadotropin surges.
The validity of this integrative model remains to be confirmed,
as does its possible importance in generating gonadotropin surges in other species.
INTRODUCTION
Neuroendocrine systems have evolved to effectively register, transduce, and integrate neural and humoral information, and to take appropriate physiological actions based
upon assessments of these incoming physiological signals.
In almost every neuroendocrine system-including the re'Correspondence: Jon E. Levine, Department of Neurobiology & Physiology, 2153 N. Campus Dr., Evanston, IL 60208. FAX: (847) 491-5211;
e-mail: [email protected]
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productive axis-classic negative feedback signals predominate in the maintenance of homeostasis. The hypothalamicpituitary-gonadal axis, however, also features a unique positive feedback control system, which is responsible for delivering a robust surge of pituitary gonadotropins into the
peripheral circulation. In spontaneous ovulators, it is this
mid-cycle, preovulatory gonadotropin surge that stimulates
the ovulatory process and has thus been a central focus of
study since the earliest years of neuroendocrine research.
The key neuroendocrine signals for stimulation of gonadotropin surges have been known for many decades, yet
the cellular mechanisms that integrate these signals are still
not well understood. It is clear that preovulatory estrogen
secretions convey signals of ovarian readiness for ovulation; in rats and probably in other species, neural signals
are also required to initiate the surge process at a specified
time and under the appropriate physiological conditions.
How are both of these necessary signals-one endocrine
(estrogen) and one neural-integrated within neurons and
pituitary cells to ultimately produce LH surges of physiological magnitude and timing? A revised model for the neuroendocrine stimulation of gonadotropin surges is discussed
below as it may apply to the female rat. The model provides
a plausible basis for neuroendocrine integrative mechanisms that mediate surge generation, and, it is hoped, represents a logical extension of concepts derived over many
years of experimentation with female rats, as summarized
below.
BASIC PHYSIOLOGY OF GONADOTROPIN
SURGES IN RATS
Secretions of LH and FSH during the ovulatory cycle of
the rat are maintained at low levels on estrus, metestrus,
diestrus, and the morning of proestrus, principally through
the negative feedback actions of gonadal steroids [1, 2] and
protein hormones, such as inhibin [3]. Under the influence
of a rising tide of estrogen secreted by the ripening follicle(s), a primary surge of both gonadotropins is released
on the afternoon of proestrus, which triggers ovulation on
the following morning of estrus. A prolonged, secondary
phase of FSH secretion continues throughout the morning
of estrus and most likely functions to recruit ovarian follicles for the subsequent cycle [4]. The secondary FSH surge
has been studied extensively, and its neuroendocrine basis
has been described elsewhere [5].
It is the positive feedback actions of estrogen, together
with the daily signal generated by the 24-h neural clock
[6], that function as the major determinants of the primary
LH and FSH surges. Ovarian progesterone released just before or during the surge greatly amplifies surges [7] and
prevents their recurrence during the same period on the next
day [8]. It is generally held that there are two critically
important, steroid-dependent processes that together mediate the LH surge-generating process: 1) hypothalamic neurosecretion of a preovulatory LHRH surge [9-11] and 2) a
coordinate increase in pituitary responsiveness to this neurosecretory trigger [12].
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HYPOTHALAMIC MECHANISMS
LHRH Pulse Generator
In virtually all physiological situations, secretions of LH
and FSH are governed by the neurosecretion of LHRH. The
LHRH decapeptide is released from neurovascular terminals in the median eminence and conveyed via the hypophysial portal vasculature to the anterior pituitary gland,
where it binds to receptors on gonadotrophs and stimulates
synthesis and secretion of LH and FSH. The LHRH release
process is almost invariably pulsatile [10-14], and in virtually all female and male mammals studied, this pulsatile
release pattern has been found to be critically important in
sustaining gonadotropin secretion [14, 15]. The cellular
mechanisms that govern the pulsatile LHRH release process, however, remain poorly understood. Electrophysiological correlates of pulse generator activity have been characterized using mediobasal hypothalamic, multi-unit recordings in monkeys [16], sheep [17], goats [18], and rats
[19]. Although these studies have not allowed for microanatomical mapping of pulsing cells and/or circuitries, they
have provided a functional definition of the "LHRH pulse
generator": a set of neurons that periodically fire a highfrequency volley of action potentials, eventuating in the
neurosecretion of a LHRH pulse into the hypophysial portal
vessels. Beyond this definition, the electrophysiological
studies have also underscored the existence of at least two
important elements of the LHRH pulse-generating process-a pacemaker, and a mechanism for electrophysiological synchronization among neurosecretory cells.
The simplest model for pulsatility holds that pacemaking
activity occurs within LHRH neurons themselves and that
the activities of "slave" LHRH neurons are entrained to
the rhythm of a dominant pacemaker within the population.
This idea is supported by the recent observations that
LHRH release from immortalized GT1 cells [20] in culture
is pulsatile [21, 22]. Pulsatile LHRH release from isolated
guinea pig mediobasal hypothalamus [23] and rat preopticmediobasal hypothalamic tissues in vitro [24] have also
been documented, and these findings, too, support the idea
of intrinsic LHRH pulsatility, since LHRH perikarya in
these species are contained within these respective tissues.
Early observations in hypothalamic-deafferented rats, however, have been difficult to reconcile with the foregoing
idea. In female rats, few if any LHRH perikarya are situated
in mediobasal hypothalamic areas, yet complete anterior
hypothalamic deafferentation does not eliminate pulsatile
LH release [25]. This paradox remains unresolved, and explanations have included possibilities that 1) descending afferent LHRH tracts may escape section in deafferentation
studies, 2) a remaining few LHRH neurons situated caudal
to sections can assume pacemaking activity, or 3) pacemaking activity is a function of cells of some other neuronal phenotype, which stimulate LHRH pulses through
synaptic contacts with LHRH processes in the median eminence. If true, the last possibility might explain the finding
that pulse generator activity can be recorded at sites within
the mediobasal hypothalamus but not at sites in the preoptic
area of the rat [19]. It is also possible that pulsatile LHRH
release occurs as an emergent property of the integrated
activities of heterogeneous neuronal populations. That there
may at least be an amplification of LHRH pulsatility by
other neurotransmitter cell groups has been suggested by
the demonstration of synchrony between LHRH pulse patterns and release profiles of other transmitters, such as nor-
epinephrine [26], neuropeptide Y (NPY) [27], and gamma
aminobutyric acid (GABA) [28].
Some mechanism must also operate to synchronize the
pulsatile release activity among LHRH neurons. Again, the
simplest scenario is one in which synchronicity is achieved
through intercellular signaling via LHRH-LHRH synaptic
contacts [29] or gap junctions [30]. Some LHRH immunopositive terminals have been demonstrated in apposition
to LHRH perikarya [29], and recent evidence suggests that
LHRH receptors are expressed in LHRH neurons [31]. Other work suggests that a volume transmission mechanism,
perhaps involving nitric oxide, could mediate synchronization among LHRH neurons [32].
LHRH Surges
Preovulatory LHRH surges have been identified in rats
[9-11], sheep [33, 34], and monkeys [35]; and indirect
measurements suggest that they may occur in women [36].
In rats, where the necessity of this neurosecretory trigger
in stimulating LH surges is unambiguous, a proestrous
surge of LHRH in this species appears to be composed of
a 2-4-h increase in the overall amount of LHRH release
[9-11].
What is the relationship, if any, between LHRH pulses
and LHRH surges? There has not been general agreement
whether LHRH surges represent an increase in some feature
of LHRH pulsatility (e.g., amplitude or frequency) or an
increase in release controlled by some nonpulsatile neurosecretory mechanism. One possibility is that signals for the
two processes are conveyed along completely separate
pathways; in this scenario, there are two populations of
LHRH neurons, with each controlling only pulsatility or
surge production. If this were indeed the case, then the
secretion of LHRH would be elevated during the LHRH
surge in a nonpulsatile manner or, at the very least, in the
absence of any change in the features of baseline LHRH
pulsatility. Alternatively, LHRH surges may occur as a result of a stimulation of LHRH pulse generator activity;
thus, a surge signal would in this case be conveyed to pacemaker cells in the LHRH pulse generator neuronal population, and an overall change in a feature of LHRH pulsatility (e.g., increased LHRH pulse amplitude or frequency)
would be manifest. In a third possible scenario, signals for
surge initiation would be conveyed to only a subset of the
pulse generator neuronal population, and not only to pacemaker cells. In such a case, a stimulatory signal would be
superimposed upon only some portion of the pulsing neuronal populations. Thus, the likely pattern of LHRH release
during the LHRH surge would consist of abrupt, transient,
and irregular changes in the features of LHRH pulsatility,
with an overall increase in the amount of LHRH released.
Our own observations in proestrous rats favor the last
possibility-that surge signals are superimposed upon the
activities of a subset of pulsing neurons. Using microdialysis to monitor LHRH release patterns at 5-min intervals,
and blood sampling through atrial catheters, we recently
re-examined the changes that occur in LHRH pulsatility as
the LHRH and LH surges proceed in proestrous or ovariectomized, steroid-primed rats [37]. As exemplified in Figure 1, the LHRH surge is characterized by acute increases
in pulse amplitude and by transient increases in pulse frequency.
Overall, the pattern appears to be much more irregular
than those in nonsurging animals, with less regular interpulse intervals and more variable pulse amplitude. Thus,
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GONADOTROPIN SURGES IN RATS
A
DAILY NEURONAL
SIGNAL
7
LHRH
PULSE GENERATOR
6
'a
5
E
*
4
3
-
2
ESTROGEN
1
0
1200
1400
1600
1800
2000
Time of Day
FIG. 1. Profile of LHRH release during a gonadotropin surge. Microdialysate samples were collected at 5-min intervals in an ovariectomized
rat treated with estradiol benzoate (30 Ig) 48 h and progesterone (5 mg)
4 h before initiation of sampling. Dialysates were analyzed for LHRH
content by LHRH RIA. Blood samples were obtained hourly via jugular
catheter, and LH levels were determined by LH RIA. Increased LHRH
pulse amplitude occurred in association with the rising phase of the LH
surge. Circles, LHRH levels in microdialysates; triangles, LH levels in peripheral plasma.
LHRH pulsatility clearly continues throughout the LHRH
surge, and the amplitude and frequency of the pulses are
increased-and more irregular-in association with the rising phase of the LH surge. These data are most compatible
with the hypothesis that neural signals for initiation of the
LHRH surge are conveyed to a subset of neurons that comprise the LHRH pulse generator (Fig. 2).
Is there a subset of "surge" LHRH neurons within the
LHRH pulse generator that may specifically function as targets for these surge signals? Lesion data indicate that the
integrity of hypothalamic sites that include the antero-ventral periventricular nucleus (AVPV) and preoptic area is
required for the elaboration of LH surges in rats. Approximately 30% of LHRH neurons, particularly those situated
in these hypothalamic areas, appear to be activated during
LHRH surges as suggested by analyses of c-fos expression
in LHRH immunopositive neurons in proestrous rats [38].
Moreover, a 25% increase in the number of preoptic neurons expressing LHRH mRNA occurs in anticipation of the
LHRH surge [39], similar to the apparent increase in the
number of LHRH immunopositive neurons at this time
[40]. It remains to be demonstrated that these subsets of
neurons share specific anatomical or electrophysiological
inputs that may convey surge-related signals.
It is interesting to note that electrophysiological correlates of pulse generator activity are diminished during the
generation of LH surges in rats [41], as they are in monkeys
[42] and goats [43]. This diminishment does not appear to
represent a desynchronization of the pulsatile activity of
FIG. 2. Schematic diagram of physiological control of LHRH surges in
female rats. A daily neuronal signal is generated by the neural clock.
Under the permissive influence of estrogen, the neuronal signal is conveyed to a subset of neurons that comprise the LHRH pulse generator.
This convergence of estrogen's permissive signals with neural signals for
the surge leads to the stimulation of subcellular processes that mediate
increased LHRH release on the afternoon of proestrus. It is not known
whether all of the neurons that comprise the LHRH pulse generator are
LHRH neurons or whether other neuronal phenotypes are also actively
engaged in pulse generation.
different units [41-43]. It is not clear how such a change
in electrophysiological activity may be associated with the
known increases (surges) in LHRH release during this period. It is possible that the apparent dissociation between
electrophysiological events and LHRH neurosecretion reflects a much greater phenotypic heterogeneity among cellular components of a broader "pulse-generating apparatus" than was previously believed. Thus, some components
of a central pulse-generating mechanism may direct release
of LHRH, and some may not, and it may be the latter units
that are suppressed during LHRH surge production. Alternatively, some acute alteration in cellular activity may occur at the level of the microelectrode recording, which may
be upstream (at or closer to the perikarya) from the cellular
locus at which pulse signals are conveyed to a given cell.
It is hoped that this interesting paradox will soon be resolved.
What Is the Daily Neuronal Signal?
In their classic 1950 study, Everett and Sawyer [6] demonstrated that injection of barbiturates during an afternoon
"critical period" in proestrous rats delays ovulation by 24
h. Their major conclusion-that a daily neural signal governs the release of the preovulatory gonadotropin surgehas been validated in numerous subsequent findings, such
as the observation that sustained estrogen treatment of ovariectomized animals produces daily, afternoon LH surges
[44]. The basic physiological control of LH surges in fe-
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male rats has thus been conceptualized as dependent upon
two phenomena: 1) generation of a daily signal within the
nervous system, probably by the circadian clock that is resident in the suprachiasmatic nucleus and 2) the positive
feedback actions of estrogen, which permit the daily neuronal signal to be conveyed to neural circuits controlling
LHRH release. This physiological control system for the
surge effectively solves two major problems in the control
of ovulation in rats: ovarian readiness and coordination
with behavior. The dependence of the surge upon estrogen
dictates that ovulation occurs only after sufficient maturation of ovarian follicles; the dependence of the surge on a
circadian, neural signal restricts its occurrence to a specific
circadian time. This restriction ensures that sexual behavior
and ovulation both occur within the same window of time,
and that both processes are initiated during the animal's
maximal waking hours.
Lesion experiments [45] and split-rhythm studies [46]
clearly demonstrate that the daily neural signal for release
of LHRH and LH surges is derived from the activity of the
circadian clock. The microanatomical substrates of the daily neuronal signal, however, are obscure and will probably
remain so until output pathways from the circadian clock
are functionally defined. It appears unlikely that a single
neurotransmitter cell group mediates transmission of the
daily neuronal signal to the LHRH releasing system, given
the sheer volume of pharmacological and transmitter effects
on preovulatory surges that have been demonstrated.
How Are Neural and Endocrine Signals Integrated in
Neurons?
The stimulation of LHRH surges occurs rapidly, suggesting that a stereotyped set of synaptic signals are the
proximal cause of surge initiation. Since estrogen treatment
(without progesterone) permits daily stimulation of LHRH
release by these signals, it is reasonable to postulate that a
major action of the steroid is to render patent the neural
pathways that convey these signals. In the absence of estrogen stimulation, these neural gates remain closed. That
estrogen acts in this manner is supported by observations
that many neurotransmitter effects on LHRH release are
maximal after animals are primed with the steroid (e.g.,
norepinephrine, NPY [47, 48]).
Few estrogen receptors have been found in LHRH neurons in the rat [49], and thus the positive feedback effects
of the steroid may be largely exerted via interneuronal networks that, in turn, control LHRH release. The neurotransmitter cell phenotypes in which estrogen may convey its
positive feedback signals may include a variety of neuronal
populations, each producing one or more amino acid,
monoaminergic, or peptidergic transmitters. The strongest
case for specific involvement of a given neurotransmitter
cell group has been made for noradrenergic, GABA-ergic,
glutaminergic, endorphinergic, and NPY-ergic neurons (see
[50] for review).
At the cellular level, it is conceivable that estrogen may
confer synaptic patency by regulating expression or activity
of one of several components of signal transduction pathways in a post-synaptic neuron, e.g., receptors, G-proteins,
amplifying enzymes, second messengers, protein kinases,
or protein kinase substrates. Indeed, in a variety of tissues
the permissive actions of estrogen have been shown to be
mediated in part by one or more of these mechanisms, one
example being the estrogen-induced up-regulation of uterine alpha-adrenergic receptor, which mediates up-regulation
of uterine contractile responses [51]. In brain, estrogen has
been shown to exert some of its effects on sexual behavior
through stimulation of oxytocin receptor gene expression
[52]. Negative effects on coupling of -adrenergic receptors
to G-proteins have also been demonstrated [53]. As yet,
however, no effects of estrogen on the expression or activity
of intracellular signaling molecules have been unequivocally demonstrated to be specifically involved in the initiation of LHRH surges.
How else may estrogen act to open neural "gates?" One
way that this might be conceptualized is that in the absence
of estrogen, i.e. in the "closed" state, a molecular component of an alternative signaling pathway is either absent
or inactive; estrogen functions to either supply or activate
that molecule and thereby permits signaling traffic through
a previously closed path. This differs from the foregoing
possibilities in that a signaling pathway is opened de novo:
signals follow a new pathway that branches off from the
cell's conventional signaling pathway. This may permit
more of an "all-or-none" gating system to operate, as opposed to a system that depends upon a graded increase in
patency of the primary signaling pathway. It would also not
require that a cell's conventional signaling pathways remain
in a functionally depressed state at virtually all times except
for the transient period following estrogen exposure. Which
molecule(s) may function as estrogen-induced, intracellular
links to pathways that mediate LHRH surge induction? Recent work in other neuroendocrine systems [54, 55] has
demonstrated that the progesterone receptor may function
in this capacity.
Progesterone Receptors and Neuroendocrine Integration
Like other members of the steroid receptor superfamily,
progesterone receptors are intracellular, ligand-inducible
transcription factors. Binding of ligand prompts a series of
events, including alterations in interactions with molecular
chaperones and dimerization of receptors, culminating in
the binding of receptors with specific cis-acting elements
of target genes. Two isoforms of the progesterone receptor,
the B form and the N-terminally truncated A form, have
been characterized, and the possibility that they mediate
different activities in cell populations is under scrutiny. Progesterone binding sites and receptor immunoreactivity have
been demonstrated in brain areas known to be important in
the regulation of LHRH surges, particularly the AVPV [56,
57]. It is not known, however, whether one of the two receptor forms is expressed to a greater extent in the AVPV
or figures more importantly in the production of LHRH
surges. In many neuronal populations, including the AVPV,
the expression of progesterone receptors is strongly dependent upon estrogen [58-61].
Treatment of estrogen-primed animals with progesterone
greatly amplifies and temporally advances gonadotropin
surges, and it is presumed that progesterone exerts these
effects through estrogen-induced progesterone receptors in
AVPV and other target neural loci. That progesterone receptor activation is critically important in the initiation of
surges is reflected by the observation that pretreatment of
proestrous rats with the progesterone receptor antagonist,
RU-486, greatly attenuates LH surges (Fig. 3; [62]).
Just before the initiation of normal preovulatory LH
surges, however, very little ovarian and/or adrenal progesterone is secreted into the circulation [63-65]. It has remained puzzling, therefore, how progesterone receptor activation may play an important role in surge initiation, at a
GONADOTROPIN SURGES IN RATS
297
12
T
saline
E
8
RU486/oil
/l
r
I
J
4
n-cci~a-/E--00
YIY-
0
1 To
I
12
14
0
l
0
I
·
·
16
18
20
s\%
02
22
TIME (hrs)
FIG. 3. Blockade of LH surges by RU-486 in proestrous rats. The type
II1progesterone receptor antagonist RU-486 (squares), or oil vehicle (circles) was injected s.c. at 1200 h proestrus. Standard error bars omitted
when smaller than corresponding symbols.
time when circulating levels of the steroid have undergone
either no change or an extremely modest rise.
On the basis of recent findings in other neuroendocrine
systems [54, 55], my colleagues and I have proposed a solution to this apparent paradox: that progesterone receptor
transactivation does not initially occur as a consequence of
hormone binding but instead results from the neural activation of intracellular second messenger systems that can
transactivate the receptor in a ligand-independent manner.
Thus, neural signals for the initiation of the LHRH surge,
e.g., the daily neuronal signal, may directly stimulate the
initial steps leading to surge production through progesterone receptor-dependent transcriptional regulation of target
genes. Two signals for surge initiation are thereby effectively integrated at the level of the progesterone receptor:
estrogen signals convey "permission" for stimulation of
surges by virtue of their ability to induce progesterone receptors; neural signals for the appropriate "timing" of the
surge are then conveyed through progesterone-independent
activation of these estrogen-induced receptors. The neural
stimulation of estrogen-induced progesterone receptors is
proposed as the initial step in a cascade of cellular events
leading to the LHRH surge. In this way, the estrogen's positive feedback signal and neural, circadian signals are integrated to produce LHRH surges of appropriate timing.
There is now considerable evidence that ligand-independent activation of steroid receptors can occur in a biological
context. Denner et al. [66] originally reported that protein
kinase (PK) A could activate avian progesterone receptormediated transcription, even in the absence of steroid ligand. Subsequent work demonstrated that the neurotransmitter dopamine can stimulate progesterone-receptor-dependent activation of transcription in transfected CV1 cells
in a ligand-independent manner [55]. The effects of dopamine appear to be mediated by PKA-dependent phosphorylation of the receptor and/or proteins that regulate DNA/
receptor binding [55]. Other examples of potential "crosstalk" between membrane- and steroid receptor-mediated
pathways include epidermal growth factor-stimulated activation of the estrogen receptor [67] and activation of the
vitamin D receptor by PKA [68]. In neuroendocrine systems, there is now compelling evidence for the physiological importance of ligand-independent activation of steroid
receptors. In pituitary gonadotrophs, the LHRH self-prim-
FIG. 4. Diagrammatic representation of model for the cellular integration of signals leading to the LHRH surge. See text for explanation.
ing process has been shown to be mediated by ligand-independent activation of progesterone receptors; that is,
LHRH-stimulated cAMP production stimulates PKA, thereby directly inducing progesterone receptor transactivation
[54]. Similarly, neurotransmitters such as dopamine appear
to facilitate sexual behavior through cAMP/PKA-mediated
activation of progesterone receptors in central neurons [55].
A Revised Model for the Mid-Cycle LHRH Surge
My colleagues and I have proposed a revised model of
the neuroendocrine processes mediating LHRH surges,
based upon the proposal that the progesterone receptor can
function as a molecular site for integration of neural and
endocrine signals. The essential components of LHRH
surge generation are schematized in Figure 4, namely, the
daily neuronal signal, estrogen positive feedback signals,
afferent neuronal circuitries, and LHRH neurons.
The daily neuronal signal is generated each afternoon,
as indicated by the arrow at the top of each panel (Fig. 4,
a-e). In the absence of a preovulatory estrogen surge (a),
no progesterone receptors are expressed in afferents to
LHRH neurons; hence, without available progesterone receptors, the daily neuronal signal is not coupled to the activation of the surge process. After preovulatory estrogen
secretion (b), the expression of progesterone receptors is
stimulated in afferent neurons, and thus (c) the daily neuronal signal, conveyed via the binding of neurotransmitters
to their receptors and production of intracellular second
messengers such as cAMP, results in ligand-independent
transactivation of progesterone receptors. Once transactivated, these receptors induce transcriptional changes in target genes, leading to increased synthesis and/or release rate
of transmitter from afferent neurons. These neural signals
are conveyed through afferent circuitries to the LHRH neuron, evoking increased synthesis and/or release of LHRH,
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which comprises the LHRH surge. The LHRH surge triggers the LH surge (d), which stimulates ovulation and secretion of ovarian progesterone. The newly secreted progesterone [65] binds to remaining, unoccupied progesterone
receptors, and their additional transactivation leads to further amplification of LHRH surges (e). With the binding
and transactivation of progesterone receptors, they are
down-regulated [69], resulting in a lack of progesterone
receptor activation by the daily neuronal signal on the next
day.
Several experimental observations on the origin of
LHRH surges may be more clearly explained by this model.
The ability of the progesterone antagonist RU-486 to almost completely block preovulatory LH surges may now
be understood as an ability of the drug to block progesterone receptor-mediated transcriptional regulation after neural
(ligand-independent) activation of the progesterone receptors. The ability of progesterone to amplify estrogen-induced LH surges and to prevent their reoccurrence on successive days may also be explained more fully: in the absence of progesterone, estrogen alone evokes small LHRH
surges, which most likely reflect only the neural activation
of progesterone receptors, without additional activation of
progesterone receptors by progesterone. Without additional
receptor occupancy-and presumably down-regulationsufficient progesterone receptors remain to permit generation of surges on successive days. If progesterone is secreted (or exogenous progesterone administered), then
many more progesterone receptors are occupied, and the
amplification of the surge occurs, as does the full downregulation of progesterone receptors [69]. The latter process
renders neurons incapable of further transmission of the
daily neuronal signal on the following day.
What other evidence is necessary to provide support for
this model? My colleagues and I have recently provided
two pieces of evidence that favor this hypothesis. In rats
that were ovariectomized on the afternoon of diestrus II,
estrogen treatments were given to stimulate LH surges on
the subsequent day. In these animals, treatment with
RU-486 was found to greatly attenuate the LH surges [70],
presumably because of the blockade of the binding of transactivated progesterone receptors to DNA. The same phenomenon was also seen in ovariectomized and adrenalectomized rats, in which progesterone secretion is presumably
nonexistent. Thus, even in the absence of progesterone as
a ligand, progesterone receptors appear to be activated by
neural signals as a requisite component of the surge-generating process. In a second study, progesterone receptor
synthesis was blocked by intraventricular application of
progesterone receptor antisense oligonucleotides. Similar to
our observations with RU-486, the antisense, but not missense oligonucleotides, greatly attenuated LH surge production in the absence of circulating progesterone [71]. In
other studies, we are currently attempting to determine
whether estrogen is likewise ineffective in stimulating preovulatory gonadotropin surges in transgenic animals lacking a functional progesterone receptor gene [72]. Beyond
these studies, proof of the validity of the proposed model
will require development of in vivo cellular markers of progesterone receptor activation. Moreover, if the model is
found to be valid, then important questions remain: Is the
progesterone receptor the only estrogen-induced transcription factor that serves as a locus for integration of neural
and endocrine signals for the surge? That RU-486 blocks
only about half of the estrogen-induced LH surge suggests
that other factors may also function as such. Is ligand-in-
dependent activation of progesterone receptors as important
in the production of LHRH surges in other species? In animals in which the LH surge appears to be less dependent
upon a circadian signal, the integration of ovarian and daily
neuronal signals may not figure as importantly. It is possible, however, that other neural command signals, not reflecting input from the circadian clock, may instead converge with ovarian steroid negative feedback at the progesterone receptor in other species.
What is the adaptive significance of this integrative
mechanism in rats? The integration of a permissive ovarian
signal (estrogen) with a timed, neural signal effectively
solves two major problems in the control of ovulation. The
permissive effects of estrogen ensure that LHRH surges
occur only after sufficient maturation of the dominant follicle, since it is only a ripened follicle that provides a sufficient estrogen surge. Without neural signals, however, estrogen alone does not initiate the surge; it merely provides
patency to neural pathways that subserve commands for
surge generation. The second adaptive advantage is derived
from the neural specification of the timing of the surge.
The dependence of surge initiation on an afternoon signal
ensures that the consequence of the surge, i.e., ovulation,
occurs coincidentally with maximal sexual receptivity and
during a circadian time of maximal wakefulness. Obviously, the greatest temporal overlap in the timing of behavioral
heat, wakefulness, and ovulation provides the greatest
chance for conception and successful reproduction.
PITUITARY MECHANISMS
The preovulatory gonadotropin surge depends also upon
the regulation of responsiveness by pituitary gonadotrophs
to the LHRH trigger. Again, both estrogen and neural signals are integrated to coordinate this process. Before the
release of the preovulatory LHRH surge, a massive, 50-fold
increase in pituitary responsiveness to LHRH occurs in two
phases. The first phase occurs gradually during the 48 h
before the surge and depends primarily upon preovulatory
estrogen secretion. An abrupt, 10-fold increase in responsiveness then occurs just before, and during initiation of,
the LH surge. The latter phase may be mediated by pituitary actions of ovarian [73] or adrenal [63] progesterone,
LHRH "self-priming" [74, 75], and the actions of additional neurohormonal "responsiveness factors," such as
NPY [76-78], galanin [79], and endothelin [80]. The delivery of the LHRH surge to this exquisitely sensitized pituitary gland results in stimulation of a preovulatory LH
surge that is 10- to 100-fold over baseline levels, and perhaps 5- to 10-fold higher than necessary to trigger ovulation
[81]. This release of excess gonadotropin during the ovulatory surge may provide insurance that the surge effectively triggers this critically important reproductive event.
The mechanisms by which estrogen may increase pituitary responsiveness have not been fully resolved but clearly involve both direct (pituitary) and indirect (hypothalamic) actions of the steroid. Estrogen's direct stimulatory effects in pituitary gonadotrophs may be exerted at numerous
points in the subcellular signaling pathways that lead to
gonadotropin release. The stimulation of LHRH receptors
by the decapeptide is now known to result in activation of
several intracellular signaling pathways, which in turn direct both early and delayed events underlying secretion of
gonadotropins. The rapid events, such as Ca 2+ entry and
exocytosis, may be mediated in part via posttranslational
protein modifications. Delayed responses, such as gonado-
GONADOTROPIN SURGES IN RATS
tropin subunit gene expression, are probably mediated by
alterations in transcription and/or translation, as well as
posttranslational modifications of proteins that participate
in signaling. It is likely that the priming of pituitary gonadotrophs by estrogen is mediated by effects of the steroid
on characteristics of both of these types of responses, including changes in gonadotropin subunit biosynthesis [82,
83], amplification of the releasable LH pool (e.g., margination of gonadotropin secretory granules) [84], and stimulation of LHRH receptor synthesis [85, 86].
Indirectly, estrogen may alter pituitary responsiveness by
inducing changes in LHRH and other neurohormone secretions, which in turn may regulate LH biosynthesis, LH secretion, or LHRH signal transduction, as discussed below.
Interestingly, the integration of estrogen-induced neurohormonal signals with estrogen's direct effects on the pituitary
gland may be mediated by ligand-independent activation of
progesterone receptors, paralleling the mechanism proposed
for central integrative processes leading to LHRH surges.
LHRH Self-Priming
The LHRH self-priming mechanism is defined as an enhanced secretory response by gonadotrophs to the second
of a pair of equal LHRH stimuli [74, 75]. The phenomenon
is estrogen-dependent, requires protein synthesis, and does
not depend upon de novo LHRH receptor synthesis [74,
75]. The process appears to be mediated, at least in part,
via the stimulation of cAMP production in gonadotrophs
[87]. It has been proposed that the self-priming mechanism
functions as an integral part of the preovulatory priming of
pituitary gonadotrophs to the decapeptide. Recent work has
shown that the progesterone receptor may function as a
critically important component of the self-priming process,
in that cross-talk may occur between cAMP/PKA and unliganded progesterone receptors [88]. Thus, LHRH may
bind receptors coupled to adenylate cyclase via Gs, and
stimulate cAMP in gonadotrophs; this may stimulate, in a
ligand-independent manner, transactivation of progesterone
receptors, which in turn regulate expression of target genes
controlling gonadotropin release.
The LHRH self-priming mechanism may essentially represent a pituitary version of the neuronal mechanism that
is proposed to integrate ovarian and neural signals for the
surge in central neurons. Similar to the proposed neuronal
mechanism, estrogen may permit the process to take place
by "seeding" pituitary gonadotrophs with progesterone receptors. Neurohormonal signals (LHRH pulses) thereafter
initiate the process at an appropriate time-just before the
surge-to increase pituitary responsiveness to LHRH. Like
the central integrative process, this mechanism provides
two major adaptive advantages for the animal: 1) it depends
upon the preovulatory estrogen surge and therefore occurs
only after presumptive follicular maturation, and 2) it is
appropriately timed by neural signals, so as to amplify surges in the afternoon hours just preceding behavioral heat.
The latter adds insurance to the likelihood that ovulation
will take place when the animal is sexually receptive.
Other Responsiveness Factors: NPY
Recent studies have confirmed that the nervous system
may produce additional neurohormones-apart from the
LHRH decapeptide-that play critically important roles in
regulating pituitary responsiveness to LHRH. In the rat, the
strongest case has been made for the 36-amino acid peptide
NPY, which is produced in greatest amounts in arcuate nu-
299
cleus neurons and released at synaptic sites throughout the
basal forebrain. The actions of NPY on LHRH release during the initiation of LHRH surges has been clearly documented [89, 90]. Importantly, NPY is also released at neurovascular terminals in the median eminence [91] and can
modulate gonadotropin secretions [76-78]. There is now a
substantial body of evidence demonstrating that NPY gene
expression [92, 93] and release [91] are increased before,
and in concert with, the stimulation of LH surges. Peripheral immunoneutralization [94] and NPY receptor blockade
[95] results in severe attenuation of LH surges, confirming
the obligatory role of this putative responsiveness factor in
the stimulation of physiologically proportioned LH surges.
The peptide has been shown to potentiate the actions of
LHRH in vitro [76, 96] and in vivo [77, 79], and, interestingly, it does not exert profound actions on its own [77].
Thus, NPY appears to subserve an important role as an
amplifier of LHRH actions. The broader, physiological significance of these actions is not clear, but NPY facilitation
may represent a permissive action by the nervous system
that amplifies the surge under conditions in which other
actions of NPY induce compatible homeostatic conditions
for reproduction, i.e., increased feeding [97] or anxiolysis
[98].
How may responsiveness factors such as NPY exert their
effects on gonadotrophs? The effects of NPY are probably
mediated via stimulation of NPY Y1 receptors [99] in gonadotrophs. NPY actions are estrogen-dependent and can
be blocked by pretreatment with RU-486 [100]. It is possible, then, that NPY may exert its effects by stimulating
the subcellular pathways that mediate LHRH self-priming.
However, NPY effects cannot be demonstrated on the
morning of proestrus, when LHRH self-priming is evident
[100]. Moreover, NPY receptors have almost always been
shown to be coupled negatively to adenylyl cyclase activity,
and not in the positive manner that would be predicted for
ligand-independent activation of progesterone receptors.
The facilitatory actions of NPY, and the steroid-dependency
of these actions, thus remain to be characterized. One result
of NPY actions on gonadotrophs is an increase in the number [101] or affinity [102] of LHRH receptors. Since unmasking of cryptic LHRH receptors has been suggested to
be mediated by PKC [103], it is conceivable that the NPY
receptor stimulation leads to phosphoinositiol hydrolysis
and PKC activation [104], and hence, augmentation of
LHRH binding site density.
In addition to NPY, there are probably other responsiveness factors that are secreted into the hypophysial portal
vasculature and can modify responsiveness of the gland to
LHRH. The peptide galanin, made in both hypothalamus
and anterior pituitary gland, may exert similar actions during the generation of preovulatory surges [79]. The importance of NPY, galanin, and other neuropeptide factors in
modulating pituitary responsiveness remains to be more
fully explored in other species, including humans.
EXTRINSIC CONTROL OF OVULATORY CYCLICITY
A variety of physiological and exteroceptive variables
exert profound influences over both the hypothalamic and
pituitary components of gonadotropin surge generation.
Neuronal systems that convey photoperiodic, stress/immune-related, appetitive, or sensory information have been
shown to regulate surge production, yet the cellular mechanisms mediating these effects remain obscure. It is not
known, for example, whether each of these regulatory in-
300
LEVINE
fluences is exerted on cyclicity upstream or downstream
from sites at which steroid and neural signals are integrated.
Indeed, the latter cell groups remain uncharacterized; while
immunocytochemical studies have revealed cell groups in
which estrogen acts to induce progesterone receptors, it has
not yet been determined which of these cells receives a
daily neuronal signal. Even more important, it remains to
be shown that ligand-independent activation of progesterone receptors occurs in these cells and that this leads to
events that produce LHRH surges.
ACKNOWLEDGMENTS
FUTURE DIRECTIONS: MOLECULAR PHYSIOLOGY OF
GONADOTROPIN SURGES
1. Gallo RV. Pulsatile LH release during periods of low-level LH secretion in the rat estrous cycle. Biol Reprod 1981; 24:771-779.
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of the pituitary gland in the same female rats. Neuroendocrinology
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the responsiveness of the anterior pituitary gland to luteinizing hormone releasing factor in the female rat. J Endocrinol 1974; 62:553572.
13. Levine JE, Duffy MT. Simultaneous measurement of luteinizing hormone releasing hormone, LH, and follicle-stimulating hormone release in intact and short-term castrate rats. Endocrinology 1988; 122:
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14. Levine JE, Bauer-Dantoin AC, Besecke LM, Conaghan LA, Legan
SJ, Meredith JM, Strobl FJ, Urban JH, Vogelsong KM, Wolfe AM.
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17. Thiery JC, Pelletier J. Multiunit activity in the anterior median eminence and adjacent areas of the hypothalamus of the ewe in relation
to LH secretion. Neuroendocrinology 1981; 32:217-224.
18. Tanaka T, Mori Y, Hoshino K. Hypothalamic GnRH pulse generator
activity during the estradiol-induced LH surge in ovariectomized
goats. Neuroendocrinology 1992; 56:641-647.
19. Kimura F, Nishihara M, Hiruma H, Funabashi T Naloxone increases
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20. Mellon PL, Windle JJ, Goldsmith PC, Padula CA, Roberts JL, Weiner
The neuroendocrine control of gonadotropin surges is a
multi-layered, temporally organized process that includes a
variety of seemingly redundant mechanisms. At the hypothalamic level, ovarian signals and directives from a neural
clock are integrated within neurons to permit stimulation
of processes leading to LHRH surges. In pituitary gonadotrophs, the actions of ovarian steroids are likewise integrated with neurosecretory signals to increase responsiveness to the LHRH surge. The net result is an appropriately
timed, explosive surge of gonadotropins that triggers ovulation on the morning of estrus. My colleagues and I have
proposed a central integrative mechanism that registers estrogen's positive feedback signals and thereafter permits
command neural signals to be conveyed. The molecular
basis of this proposed mechanism is the ligand-independent
activation of progesterone receptors. A similar mechanism
has already been proposed as the basis of estrogen's permissive actions with respect to the self-priming effects of
LHRH in pituitary gonadotrophs. Confirmation of these hypotheses will probably require development of new in vivo
models, such as transgenic animals in which the activation
of progesterone receptors can be marked in individual cells
in the brain. It also remains to be determined whether progesterone is but one of several transcription factors induced
by estrogen and subsequently activated by neural signals.
In 1983, it was demonstrated that few estrogen receptors
are expressed in LHRH neurons. Since that time, intensive
searches have been underway by numerous laboratories to
identify the neuronal phenotypes that may convey positive
feedback effects of estrogen. An argument can now be
made that we are perhaps victims of our own success. Virtually every neurotransmitter imaginable has been shown
to be either regulated by estrogen, or able to regulate LHRH
release, or both. Countless regulatory schemes have been
formulated accordingly, to describe how cells may interact
to stimulate the LHRH and LH surges. It is arguable, however, whether the sum of the parts of these schemes has
yielded a clearer picture of how the whole system operates.
The urge to construct intricate circuitries that control preovulatory LHRH release, based upon supposedly uniform
actions of various transmitter cells, has perhaps obscured
an even more important issue: how does physiological integration of neural and endocrine signals take place at the
molecular level, in any cell? When we are better armed
with this knowledge, it may then be a less daunting task to
consider which neuronal populations may serve in this capacity. The hypotheses proposed in this paper may ultimately be proven valid, partially valid, or untenable. At the
very least, however, it is hoped that they provoke a significant change in the prevailing paradigm, namely, in how
we conceptualize and experimentally approach the cellular
basis of the preovulatory gonadotropin surge.
This work was supported in part by NIH grants R01-HD20677, P01HD21921, and P30-HD28048. Many of the trainees who participated in
the studies cited were supported by a Training Grant in Reproductive Biology from the National Institute for Child Health and Human Development, T32-HD07068. I am grateful to my colleague, Dr. Janice H. Urban,
and to Patrick E. Chappell, Sarah M. Leupen, and Ming Xu for their help
and advice in the writing of this review.
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