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Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208
Address all correspondence and requests for reprints to: Jon E. Levine, Ph.D., Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208. E-mail: jlevine{at}nwu.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In these studies and in a companion paper, we tested the hypothesis that coupling of the neural surge signal to the GnRH neurosecretory process is mediated by an essential, two-step process: 1) E2 induces expression of intracellular, transcriptional regulators in target hypothalamic neurons; and 2) neural signals for the surge subsequently activate these transcriptional regulators, prompting alterations in gene expression that are necessary for initiation of the GnRH surge. Our experiments focus specifically on the idea that progesterone receptors (PRs), particularly those expressed within neurons in or near the anteroventral periventricular nucleus (AVPV), may function as E2-induced transcriptional regulators in this proposed integrative mechanism. A priori, there are several reasons to suspect that hypothalamic PRs function in this manner: hypothalamic PR expression is induced by E2 (4, 5, 6), PR activation results in advancement and amplification of GnRH (7) and LH surges (8, 9), and recent studies have indicated that PRs can be activated by neural signals (10, 11), presumably in a ligand-independent manner (11, 12). The PRs in the AVPV have been specifically implicated in mediating E2-induced GnRH surges; after approximately 24-h exposure to high titers of E2, an abundance of both PR mRNA (6, 13) and protein (5) is observable in the AVPV in female rats. Ablation studies, moreover, have demonstrated that the AVPV represents a region that functions as an indispensable part of the LH surge timing mechanism (14).
It has not been determined, however, whether activation of these E2-induced PRs in the AVPV or adjacent areas is an obligatory step in the stimulation of GnRH surges by E2. Our recent work in PR knockout (PRKO) mice has confirmed that activation of some population of PRs is requisite in the production of LH surges (15). In that study, however, the impact of hypothalamic PR ablation on GnRH surges could not be differentiated from the demonstrated involvement of pituitary PRs in GnRH self-priming and LH surge generation. The present studies were thus designed to specifically assess the role of hypothalamic PRs in the generation of GnRH surges. In one set of experiments, microdialysis procedures were used to monitor GnRH release and to determine whether systemic administration of a PR antagonist blocks E2-induced GnRH surges. In the second set of experiments, PR antisense oligonucleotides were administered intracerebroventricularly (icv) to determine specifically whether activation of PRs in the AVPV and adjacent regions is an absolute requirement for the generation of GnRH and LH surges. Our findings demonstrate that activation of PRs in the AVPV plays an obligatory role in the stimulation of GnRH and LH surges by E2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Exp 1: effects of PR antagonist on GnRH release. On the day following E2B-priming, rats received a sc injection of ZK98299 (6 mg/kg in benzoyl benzoate/sesame oil) or vehicle at 1000 h. At 1200 h, microdialysis probes (CMA/12; CMA/Microdialysis) were inserted into guide cannulas such that the semipermeable membrane at the tip was positioned at the level of the lateral median eminence. Artificial cerebrospinal fluid was pumped through probes via a syringe pump (CMA/100; CMA/Microdialysis) for an initial 60 min equilibration period at a flow rate of 2.0 µl/min. From 1300 h to 2000 h, dialysate samples were collected at 5 min intervals into borosilicate glass tubes containing RIA buffer (PBS containing 1% gelatin), immediately snap-frozen in a dry ice-ethanol bath, and stored at -80 C for subsequent GnRH RIA. During microdialysis sessions, 0.25 ml blood samples were withdrawn from the atrial catheters every hour, followed by replacement of equal volumes of 0.9% saline, until 2000 h. Samples were centrifuged at 4 C, and plasma was stored at -20 C for LH RIA. After completion of experiments, rats were anesthetized and then killed by exsanguination. Brains were frozen on dry ice, and stored for subsequent sectioning and histological verification of cannula placement. Histology revealed a high precision of cannula placement, with a rostro-caudal range of 200 µm, and a mediolateral range of 50 µm.
Exp 2: effects of icv PR antisense oligonucleotide infusions on LH surges. Antisense, sense, and missense oligos were synthesized as phosphorothioated 20-mer. The antisense oligos were designed to be complimentary to the region of the translation start site of the PRA isoform of the receptor (5'-GCTCATGAGCGGGGACAACA-3'). Sequences for sense and missense control oligos were 5'-TGTTGTCCCCGCTCATGAGC-3' and 5'-TGTTAAAGGTCAGGAATAGC-3', respectively. Oligo sequences were verified by GenBank to preclude possible complimentarity to other eukaryotic gene sequences. Oligos were dissolved in 0.9% saline to a final concentration of 4 nM, and 1 µl was injected into the 3V for 1 min via a 33-gauge injection cannula connected to a 10-µl Hamilton syringe (Reno, NV). Animals were infused with oligos or saline at 1800 h on day 1 and at 0600 h on the day after E2B injection (day 2). This oligonucleotide infusion regimen is similar to others used to prevent expression of PR in the ventromedial nucleus (16). A similarly cannulated cohort of ovary-intact animals was treated with saline only for comparison of progesterone (P) levels with OVX, E2B-primed saline-treated rats. Blood samples were withdrawn from atrial catheters hourly, beginning at 1200 h and ending at 2200 h on day 2. The samples were centrifuged, and plasma was stored at -20 C for later RIA. Confirmation of cannula placement was assessed as described above.
Immunocytochemistry
One cohort of OVX, E2-primed antisense-,
missense-, and saline-treated animals was used for immunocytochemical
confirmation of antisense effectiveness in preventing expression of PR.
Animals were given 75 mg/kg pentobarbital (Sigma, St.
Louis, MO), ip, at 1500 h on day 2 and transcardially perfused
with saline containing 2% sodium nitrite (Sigma) followed
by 5% acrolein (Polysciences, Warrington, PA) and 4% paraformaldehyde
(Sigma). Brains were removed, blocked, postfixed for
2 h in 4% paraformaldehyde, and stored overnight in 20% sucrose
at 4 C. Brains were then sliced on a cryotome at -18 C, and 30-µm
slices were placed in a cryoprotectant solution at -20 C for storage.
Sections were then rinsed in 0.05 M Tris-buffered
saline (TBS), pretreated with NaBH4, and
rinsed in TBS again. Nonspecific binding was reduced by briefly
incubating slices in TBS containing 1% hydrogen peroxide and 5%
normal goat serum. Sections were then incubated for 72 h in a
1:20,000 dilution of an anti-PR antibody, directed against the
DNA-binding domain of human PR (533–547, DAKO Corp.,
Carpenteria, CA) at 4 C. The primary antiserum was omitted for control
sections. After rinsing, slices were incubated in a biotinylated
secondary antibody (antirabbit IgG) followed by incubation in AB
reagents for 90 min each (Vector Laboratories, Inc.,
Burlingame, CA). After TBS rinses, sections were placed in a 0.05%
3,3-diaminobenzidine-HCl (Sigma) solution for 10 min,
transferred to subbed slides, allowed to dry, and coverslipped.
Sections were examined from the organum vasculosum of the lamina
terminalis caudal to the mammilary bodies. Cells staining for PR were
counted only within a 1.0-mm wide region centered on the midline from
the ventral surface of the brain to the dorsal aspect of the 3V.
Numbers of PR-immunoreactive (PR-ir) cells were counted and compared
with atlas-matched regions in each treatment group.
RIAs
LH standard (RP-3) was provided by NIDDK. The sensitivity
of the LH RIA was 40 pg/tube. The GnRH antibody EL-14 was provided by
Dr. Martin Kelly (Oregon Health Sciences University, Portland,
OR). The sensitivity of GnRH assay was 0.1 pg/tube. Intraassay
coefficients of variance for LH and GnRH were 8.6% and 10.2%,
respectively. P was assayed using the Immuchem Prog
125I kit (ICN Pharmaceuticals, Inc.,
Costa Mesa, CA). The intraassay coefficient of variance for P was
5.6%
Statistical analyses
Significant GnRH pulses were determined using the ULTRA pulse
analysis program (17). Plasma LH and dialysate GnRH data are presented
as representative release profiles in the experiments using the
antiprogestin ZK98299. In the experiments involving antisense
oligonucleotide treatment, LH data are presented as the mean ±
SEM. Comparison of oligonucleotide treatment groups across
time as well as comparison of plasma P levels in OVX,
E2B-primed vs. intact animals were
carried out using a two-way ANOVA with repeated measures, followed by
Bonferroni’s post-hoc test. Comparison of mean GnRH
release, mean pulse amplitude, and mean pulse frequency before and
after 1600 h in both antiprogestin-treated and control groups was
performed using a paired one-way ANOVA. Differences in PR-ir cell
number due to oligonucleotide treatment within each rostrocaudal range
were calculated using a paired one-way ANOVA. In all analyses,
P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, OVX
rats that were primed with E2B exhibited
significant increases in GnRH pulse amplitude (Fig. 2A) between 1600–2000 h compared with
pulses measured before 1600 h in the same animal. Mean GnRH pulse
frequency, however, remained unchanged throughout the sampling period
(Fig. 2B). Mean GnRH release among E2B-primed
rats between 1600–2000 h increased about 3- to 4-fold compared with
GnRH release before 1600 h (Fig. 2C). Additionally,
E2B-primed, OVX rats exhibited LH surges between
1600–2000 h that followed GnRH release patterns closely, demonstrating
a 3- to 5-fold increase in LH levels compared with those in untreated
OVX controls (Fig. 1A).
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) exhibited
no significant increases in mean GnRH pulse amplitude (Fig. 2A),
frequency (Fig. 2B), or release (Fig. 2C) from 1600–2000 h compared
with these parameters between 1200–1600 h. Moreover, no significant
increase in plasma LH was observed in these animals throughout the
sampling period. Plasma LH levels in rats treated with the
antiprogestin resembled those observed in OVX rats not primed with
E2B (data not shown).
Antisense oligonucleotide treatments
Effects of antisense oligonucleotides on LH surges. Acutely
OVX rats primed with E2B on day 1 and
administered saline icv exhibited plasma LH surges occurring between
1700–2000 h on day 2. In contrast, rats infused icv with PR antisense
oligonucleotides exhibited no elevation in plasma LH during the same
sampling period (Fig. 3A). Controls
treated with missense and sense oligonucleotides icv exhibited plasma
LH elevations similar to those observed in saline-treated controls
(Fig. 3, B and C). As noted in earlier studies (8), LH surges in OVX,
E2-treated rats are more variable in timing and
magnitude than those previously observed in proestrous animals or in
OVX animals additionally receiving P. In some cases, surges were
irregular in form, reaching more than one major apex. Overall, however,
there were no discernible differences in the amplitude or timing of the
LH profiles among E2-primed animals undergoing
microdialysis, receiving icv injections, or receiving no stereotaxic
implants (data not shown). OVX animals not treated with
E2B showed no increase in LH release regardless
of oligonucleotide treatment, and there was no significant effect of
any oligonucleotide treatment on basal LH levels in these animals (data
not shown).
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and 5 and Table 1). Although PR staining was
significantly decreased in antisense oligonucleotide-treated animals
compared with that in saline- and missense-treated animals in the AVPV
and medial preoptic nucleus, the number of PR-ir cells examined
further caudally, particularly in the arcuate nucleus and ventromedial
hypothalamus (VMH), was not significantly different from that in
saline- or missense-treated controls, suggesting that the region of
effectiveness of the antisense the oligonucleotides was limited to a
narrow rostrocaudal range (Figs. 4 and 5).
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). OVX, E2B-primed
rats exhibited increases in LH release (Fig. 3A) even in the absence of
any increase in circulating plasma P. P levels in ovary-intact animals,
however, increased only after the ascending phase of the LH surge,
suggesting that the major rise in P observed on proestrus occurs
subsequent to LH surge release. Interestingly, P levels in both
treatment groups declined by about 50% before the start of LH surges.
Although it remains unclear why this occurs, this observation is in
accordance with earlier studies (19). Regardless of this initial
decrement in circulating P, it is clear that P levels do not exhibit
any increase before LH surges in either OVX or intact animals and are,
in fact, decreased just before the ascending phase of the surge.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the first of these studies, E2B treatments were found to stimulate coincident GnRH and LH surges in acutely OVX rats. Additional administration of the type I antiprogestin ZK98299, however, leads to a complete blockade of E2B-induced GnRH and LH surges. This type I PR antagonist, after binding to the receptor, has been shown to prevent conformational changes necessary for DNA binding, thereby eliminating the possibility of the antiprogestin acting as a partial PR agonist (22). The most straightforward interpretation of this pharmacological result thus holds that the activation of some neuronal PR population is a necessary event in the stimulation of GnRH surges by E2.
In the second set of experiments, we assessed whether the AVPV is
a specific neural locus at which PR activation functions as a critical
step in GnRH surge generation. Evidence favoring the AVPV as such an
integrative site is abundant; the area is a sexually dimorphic region,
and it expresses high levels of ER
and some ERß as well as
E2-inducible PRs (13, 23). Ablation or
isolation of the AVPV prevents gonadotropin surges regardless of
E2 and/or P treatment (14, 24). Suprachiasmatic
nuclei (SCN) ablations also prevent LH surges in intact or
E2-primed animals; however, LH surges can be
elicited with E2 plus P (14). These studies
suggest that whereas the SCN provides the daily signal necessary for
initiation of GnRH surges, the AVPV acts as an integrative center where
this afferent signal can be converted into a GnRH surge signal under
the permissive effects of E2. Supporting this
idea are recent studies that have shown that AVPV neurons receive
signals originating in the SCN, predominantly on ER-expressing cells
(25), and that certain ER-positive AVPV neurons send efferents that
form synapses with GnRH perikarya in the medial preoptic area (26).
Other work has demonstrated that the ER antagonist keoxifine,
administered into this region, is sufficient to block LH surges (27).
We have found that infusion of PR antisense, but not missense or sense
oligonucleotides, in the ventricular spaces adjacent to the AVPV
resulted in complete blockade of LH surges. Although we could not
measure GnRH release directly in our second experiment, it is
reasonable to assume that LH surges were prevented as a result of the
blockade of antecedent GnRH surges. The results of our PR
immunohistochemical studies provide direct evidence for this
contention, as the same antisense treatments, but not missense or sense
oligonucleotide treatments, produced precipitous declines in PR
expression only in a confined area proximal to the injection site, just
caudal to the organum vasculosum of the lamina terminalis. The PR
immunoreactivity in more caudal regions, by contrast, was not
attenuated. It must therefore be concluded that blockade of PR
expression in the AVPV and adjacent regions is sufficient to prevent
the initiation of GnRH and LH surges. Taken together, the results of
both experiments demonstrate that hypothalamic PR activation is
obligatory in the stimulation of GnRH surges by
E2, and that the PRs expressed in the AVPV and
adjacent regions are especially important in this process.
It has been known since the classic experiments of Everett et al. (8, 28) that treatment of E2-primed female rats with P can temporally advance and greatly amplify gonadotropin surges. That PR activation is critically important in the production of preovulatory hormone surges is reflected by the observation that pretreatment of female rats on proestrus with PR antagonists results in blockade of LH surges (29, 30). Just before the initiation of preovulatory gonadotropin surges, however, very little ovarian and/or adrenal P is secreted into the circulation (31, 32). It has thus been difficult to understand how PR activation may play an important role in surge initiation at a time when circulating levels of P have undergone either no change (33, 34) or an extremely modest rise (35). As a solution to this apparent paradox, we recently proposed that PR trans-activation may initially be stimulated by a neural signal, independent of changes in circulating P (2). Our current results support this concept, as PR antagonism was found to block E2B-induced GnRH surges even in the absence of any changes in P in the circulation of these animals. Indeed, we found that levels of P in OVX, E2B-primed rats were significantly lower than those concentrations observed in ovary-intact rats at all time points throughout proestrus. Additionally, no increase in P was observed throughout the sampling period in OVX, E2B-primed rats, in agreement with the hypothesis that hypothalamic PRs may be activated independently of a change in circulating ligand.
Through what mechanism, then, could brain PRs be activated by neural signals, independent of signal changes in circulating P? One route is through enhanced production of P in brain (36), and subsequent activation of brain PRs by this locally produced neurosteroid. Measurements of P in brain during the estrous cycle, however, do not support this idea, as P levels have been found to reach their nadir on the afternoon of proestrus (37). A second possibility is that neural signals activate PRs in a ligand-independent manner (12, 38). There is now considerable evidence that ligand-independent activation of steroid receptors can occur in a physiological context. In pituitary gonadotropes, for example, the GnRH self-priming process has been shown to be mediated by ligand-independent activation of the PR (39); the sequence of signaling events appears to include GnRH-stimulated cAMP production and activation of protein kinase A (PKA), leading to PR trans-activation in the absence of P. Our recent finding that GnRH self-priming does not occur in PRKO mice (15) lends support for the importance of this signaling pathway. Similarly, neurotransmitters, such as dopamine, have been shown to facilitate sexual behavior through cAMP/PKA-mediated activation of PRs in central neurons (11). A companion paper addresses more directly the hypothesis that ligand-independent activation of PRs, specifically by a cAMP/PKA-mediated pathway, mediates the neural activation of PRs that prompts the release of GnRH surges (40).
Our findings demonstrate that neuronal PR activation is a necessary component of the processes leading to GnRH surges and suggest that the initial activation of PRs occurs through a mechanism independent of peripheral changes in P concentrations. After initiation of the LH surge, however, a robust and prolonged surge of P is released. What are the biological functions of this proestrous P surge? The ability of P to augment GnRH and LH surges is most likely indicative of an amplification function of the P surge during the spontaneous GnRH and LH surges on proestrus (41, 42). Thus, P released during the rising phase of the GnRH and LH surges may continue to prompt trans-activation of PRs through a ligand-dependent process and thereby exert its well known amplifying effects. In this manner, the neural signals direct the initiation and, hence, appropriate timing of the GnRH surge, whereas P enhances the proportions of the GnRH and LH surges, possibly as added insurance that ovulations are successfully triggered.
Earlier studies demonstrated that the proestrous surge of P also functions to prevent the release of LH surges on subsequent days. Thus, administration of P to animals receiving prolonged E2 treatments leads to an amplification of the LH surge on the day that P is administered and an extinction of the LH surge on the following afternoon (43). Previous studies have demonstrated that P treatments can down-regulate the expression of PR mRNA (44, 45) as well as PR protein (46). If the proestrous P surge leads to a down-regulation of PRs, then this may provide a cellular basis for the ability of P to extinguish LH surges; that is, as we have demonstrated that PR activation is obligatory in the stimulation of GnRH surges, it would be predicted that sufficient down-regulation of PRs would be accompanied by a refractoriness to E2 positive feedback effects. Thus, the relative depletion of PR receptors by a P surge would be expected to produce an uncoupling of the neural signals for the GnRH surge from the GnRH neurosecretory system. The ability of P to extinguish LH surges on the day after P release may therefore be mediated by the ability of the steroid to down-regulate its receptor in the AVPV.
During the estrous cycle of the rat, the preovulatory E2 surge conveys a permissive, yet essential, signal representing the readiness of ovarian follicles for an ovulatory stimulus. The consequence of this permissive signal is the coupling of neural signals for release of GnRH surges to the neural circuitries governing GnRH release; the timing of the GnRH surge is thereafter dictated by the temporal characteristics of the daily neural signal. We hypothesized that the expression and activation of PRs are obligatory steps in this E2-dependent coupling process. Our working model holds that E2 confers patency to the signaling pathways leading to GnRH surges by virtue of its ability to induce PR expression in the AVPV; initiation of GnRH surges thereafter occurs through neural activation of these PRs, and subsequent PR-induced transcriptional responses mediating the release of GnRH surges. In these experiments, we have obtained direct support for the validity of one feature of this hypothesis, viz. that PR activation in the AVPV is an obligatory event in the stimulation of GnRH surges by E2. A second major feature of our model, that PRs in the AVPV can be activated by specific intracellular signaling components, is assessed in the companion paper that follows this report (40).
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Acknowledgments |
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Received October 4, 1999.
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