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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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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These experiments tested the specific hypothesis that intracellular cAMP mediates the daily neural signal that trans-activates PRs in the AVPV, thereby prompting GnRH surges. Recent in vitro studies have demonstrated that cAMP can activate chicken, mouse, and human PR in the absence of progesterone (P) (5, 6) as well as potentiate agonist-mediated increases in transcriptional activity in a cell- and promoter-specific manner (7). Activation of either the A or B isoform of human PR can stimulate transcription in the presence of cAMP and in the absence of ligand (8). This second messenger has also been implicated in ligand-independent activation of PRs that appears to mediate GnRH self-priming (6) and the facilitation of sexual behavior by dopamine (DA) (9).
Based on the foregoing evidence, we sought to determine whether cAMP manipulations in AVPV neurons result in alterations of LH surge release, and if any such effects are mediated by PR activation. Our model predicts that 1) cAMP levels in the AVPV are elevated just before GnRH and LH surges; 2) cAMP levels are similarly increased at the same time on all other days; 3) premature elevations of cAMP in the AVPV advance LH surges, whereas blockade of the endogenous cAMP signal prevents surges; 4) both normal and cAMP-advanced surges are blocked by PR antagonism; and 5) P administration can rescue surge release after blockade of the cAMP signal. Experiments were thus designed to test each of these predictions, to thereby fully assess whether cAMP functions as a major component of the daily neural signal governing the release of E2-induced GnRH surges.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Measurement of cAMP levels in brain tissues
Proestrous, estrous, and metestrous rats were killed via
decapitation at 0900, 1200, 1500h, 1800, and 2200 h. Additional
groups of rats were ovariectomized (OVX) under methoxyfluorane
anesthesia via bilateral abdominal incision. The latter rats were
treated 5 days later with either estradiol benzoate
(E2B; Sigma, St. Louis, MO) or oil
vehicle, sc, and were killed by decapitation on the following day, at
the same time points as the ovary-intact animals. Whole brains were
removed and immediately fresh-frozen on dry ice. The brain tissues were
blocked and mounted in a cryotome, and 40-µm slices were removed
until the caudal-most portion of the organum vasculosum of the lamina
terminalis was exposed. At this point, small incisions were made
with an ultrafine scalpel blade (Fisher Scientific,
Pittsburgh, PA), defining a 1.0-mm wide region centered at the midline,
and extending 2.5 mm from the ventral surface. A 1.0-mm thick tissue
slice was then removed using the cryotome blade. Sections of equal
dimensions were excised from the ventromedial hypothalamus (VMH) and
the frontoparietal motor cortex to serve as controls. All sections were
homogenized immediately in a Teflon-glass homogenizer on ice in a
volume of 0.4 ml acid-ethanol solution. Fifty microliters of homogenate
were removed, and protein content was determined by Bradford assay.
Homogenates were centrifuged at 1000 x g for 20 min at
0 C. Supernatants were removed, placed into polypropylene tubes, and
lyophilized in a Speed-Vac evaporator (SVC200H, Savant Instruments,
Farmingdale, NY) for 8 h. Pellets were resuspended in 50
mM Tris-HCl assay buffer containing 4
mM EDTA and 100 µM
isobutylmethylxanthine (Sigma) to prevent cAMP
degradation. cAMP measurements were determined via competition with 5.0
nM [8-3H]cAMP
(Amersham Pharmacia Biotech, Piscataway, NJ) using a
binding protein isolated from bovine adrenals. Standard curves were
generated for each assay, protein-bound
[8-3H]cAMP was placed in ScintiVerse
(Fisher Scientific) fluid, and radioactivity was
determined by a Beckman Coulter, Inc., liquid
scintillation counter (Palo Alto, CA).
Surgical protocols and hormone treatments
Rats were anesthetized with 10 mg/kg ketamine (Ketaset, Fort
Dodge Laboratories, Fort Dodge, IA), ip, and 8 mg/kg xylazine (Gemini
SA, Burns Veterinary Supply, Inc., Rockville Center, NY), im, and
implanted with single barrel guide cannula (Plastics One, Roanoke, VA)
stereotaxically directed to the rostral-most portion of the third
ventricle (0.5 mm caudal to bregma; 8.0 mm ventral to the skull).
During cannula insertion, the sagittal sinus was moved laterally using
a blunted 27-gauge needle to minimize bleeding. An additional cohort of
animals was implanted with cannulas directed 2 mm caudal to determine
whether treatments distal to the AVPV could have similar effects. After
resumption of estrous cyclicity, rats were anesthetized with
methoxyfluoane (Metofane, Pittman-Moore, Inc., Washington Crossing,
NJ), OVX at 0900 h on diestrous day 2 (day 1), given a sc
injection of E2B (30 µg), and fitted with
indwelling atrial catheters (PE-50, Becton Dickinson and Co., Parsippany, NJ) inserted through the jugular vein and
exteriorized at the nape of the neck. Stainless steel plugs were
inserted into the free end of the catheters to occlude them until
sampling on the following day.
Intracerebroventricular (icv) 8-bromo-cAMP administration
On the day after E2B priming, rats
received an icv infusion of 8-bromo-cAMP (Sigma, St.
Louis, MO) or saline at 0900 h. The 8-Br-cAMP was dissolved in
0.9% saline to a final concentration of 10 mM, and 1.0
µl of this solution was injected icv over 2 min via a 33-gauge
injection cannula connected to a 10-µl Hamilton syringe (Reno, NV).
One cohort of 8-bromo-cAMP-treated rats received sc injections of 6
mg/kg RU486 (a gift from Roussel-UCLAF, Romainville, France) at
1000 h, and the rest received sesame oil vehicle. Blood samples
(0.25 ml) were collected half-hourly from 0830–1000 h and then hourly
until 2100 h, and centrifuged at 4 C. Replacement of equal volumes
of 0.9% saline followed the withdrawal of each sample. Plasma was
removed and stored at -20 C for LH RIA. Rats were killed the following
day, brains were fresh-frozen on dry ice, and histochemical staining
was performed for verification of cannula placement. Briefly, 40-µm
sections were sliced on a cryotome, thaw-mounted on glass slides,
stained with 0.8% cresyl violet, dehydrated with increasing
concentrations of ethanol, and coverslipped.
ICV SQ 22536 administration
An adenylyl cyclase inhibitor, SQ 22536
[9-(tetrahydro-2'-furyl)adenine] (Calbiochem, San Diego,
CA), was dissolved in 0.9% saline to a final concentration of 10
µM and infused as described above at 1100 h on the
day after E2B injection. One cohort of
E2B-primed, SQ 22536-treated animals was also
given P (8 mg/kg in sesame oil; Sigma) or oil vehicle at
1000 h. Blood samples were withdrawn from atrial catheters hourly,
beginning at 1200 h and ending at 2200 h. Blood samples were
centrifuged, and plasma was removed and stored at -20 C for later LH
RIA, and cannula placement was assessed histologically as described
above.
RIAs
LH standard (RP-3) was generously provided by NIDDK. The
sensitivity of the LH RIA was 40 pg/tube. The intraassay coefficient of
variance for LH was 12.2%. LH and cAMP data are presented as the
mean ± SEM. cAMP levels were normalized to the
protein content of homogenates and presented as picomoles per mg
protein.
Statistical analysis
The mean cAMP content measured from AVPV homogenates at
different sacrifice times was compared with levels at the 0900 h
point via ANOVA, with P < 0.05 considered significant.
Data obtained from treatment groups in the 8-Br-cAMP and SQ 22536
experiments were compared using a two-way ANOVA with repeated measures,
followed by Bonferroni’s post-hoc tests, with
P < 0.05 considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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as
picomoles per mg protein. ANOVA and post-hoc comparisons
revealed a significant (P < 0.05) peak at the
1500 h point compared with the 0900 h point. Lowest levels of
cAMP in the AVPV were observed at 1800 h. These patterns of
intracellular cAMP accumulation, viz. significantly
increased at 1500 h, were also evident in the data from rats
killed on estrus (Fig. 1B) and metestrus (Fig. 1C) as well as in 5-day
OVX rats (Fig. 1D) and 5-day OVX rats given E2B
(Fig. 1E). Tissue blocks containing the AVPV were cut just caudal to
the organum vasculosum of the lamina terminalis and extend 1 mm
caudally. Although it is possible that these hypothalamic sections
contain cells from the rostral SCN, it is unlikely that the cAMP rhythm
observed in this study represents oscillations in the SCN, as a
previous study revealed a different pattern of cAMP
accumulation in this region (10). Comparison of cAMP content in VMH
(Fig. 2A) and motor cortex (Fig. 2B) among the same time points on proestrus and estrus revealed no such
increase at 1500 h. An observable, yet not statistically
significant, peak in VMH cAMP content was observed at 1200 h on
estrus and may represent an increase in neuronal activity associated
with mating behavior, although this is not clear from the present
studies. cAMP levels in the motor cortex exhibited no significant
differences among all time points.
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). In OVX rats that
were not treated with E2B, no elevations in
plasma LH were observed. In these unprimed animals, moreover,
8-bromo-cAMP infusions were entirely ineffective in altering LH
secretion.
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Effects of adenylyl cyclase inhibition on LH surges
Similar to the previous experiment, OVX,
E2-treated rats given saline icv exhibited plasma
LH surges beginning at 1600 h and remaining elevated until
2100 h. In contrast, OVX, E2-treated rats
receiving infusions of the adenylyl cyclase inhibitor SQ22536 icv at
the level of the AVPV exhibited no elevation in plasma LH
(P < 0.05) compared with saline-treated controls (Fig. 4). Animals in which the inhibitor was
infused 2 mm caudal of the AVPV, however, demonstrated no significant
attenuation in LH surge levels compared with OVX,
E2-primed controls infused with saline,
suggesting that adenylyl cyclase inhibition only in the AVPV and
rostral periventricular regions prevents GnRH surge generation. P
treatment of OVX, E2-primed rats given SQ22536
icv, however, effectively restored LH surges. In these animals, LH
surges were observed, which were significantly (P <
0.05) advanced and augmented compared with LH surges in
E2-primed rats treated with saline icv (Fig. 4).
Animals primed with E2B and P in the absence of
SQ22536 treatment exhibited similar LH surge profiles as those given
the inhibitor (data not shown), suggesting that P administration can
initiate LH surge generation on proestrus in the presence or absence of
adenylyl cyclase activity in the AVPV.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One major prediction of our model is that cAMP levels in the AVPV are elevated on a daily basis, just before the initiation of the GnRH surge. Our measurements of GnRH release profiles in E2-treated rats previously showed that the initiation of GnRH surges occurs between 1500–1600 h. Thus, the finding that cAMP levels are elevated at 1500 h in the AVPV, but not in control regions, directly supports the idea that this rise in cAMP represents a timed neural signal leading to the surge. That a similar elevation was seen in rats on all days of the cycle and in OVX rats regardless of E2 treatments additionally indicates that this signal is a daily one, probably stimulated by afferent impulses derived from the 24-h biological clock. Further studies are required, however, to determine in what cell type(s) this intracellular accumulation of cAMP occurs, to more definitively implicate this increase as playing a causative role in GnRH surge release.
Our integrative model for the surge holds that neural signals for the surge stimulate GnRH surges via neural activation of PRs. Accordingly, premature delivery of a signal for the surge would be expected to advance the surge, whereas blockade of the endogenous neural signal should prevent its release. If cAMP in the AVPV comprises this daily neural signal for the surge, then by this reasoning it would be expected that cAMP elevations induced before the onset of an endogenous cAMP rise would temporally advance GnRH surges, whereas blockade of cAMP production would prevent GnRH surges. The outcomes of our experiments fulfill both of these expectations; icv infusion of the cell-permeant cAMP analog, 8-bromo-cAMP, was found to advance the initiation of LH surges, whereas infusion of an adenylyl cyclase inhibitor completely blocked surge release. These observations additionally support the idea that cAMP elevations in the AVPV function as a manifestation of the daily neural signal governing the release of GnRH surges.
It should be noted, however, that we could not directly measure GnRH
release in experiments in which icv infusions were administered;
instead, we made the assumption that the temporal characteristics of
the LH surge faithfully represented the timing of the underlying GnRH
surge in these animals. In the previous study, a close temporal
association was demonstrated between the rising phases of GnRH and LH
surges, and thus the presumption of such a relationship in these
experiments would appear to be a reasonable one. The specific targeting
of the icv pharmacological treatments to the AVPV and adjacent regions
was also an important consideration in these experiments, as it was
possible that significant concentrations of the administered drugs
could have been transported to more caudal hypothalamic regions or to
the pituitary gland via the portal vasculature. Infusions of SQ 22536
into more caudal ventricular positions, however, were ineffective in
preventing the release of estrogen-primed LH surges, making it unlikely
that any observed effects of pharmacological treatments resulted from
actions at more caudal sites. An additional possibility is that
8-bromo-cAMP or SQ 22536 may have diffused more laterally and altered
the activity of the cAMP/protein kinase A pathway in GnRH cell bodies
themselves. This possibility seems unlikely considering that the
response to 8-bromo-cAMP was seen only in
E2-primed animals despite the fact that GnRH
neurons in rats express few, if any, E2 receptors
in female rats (11). Although a recent study demonstrates that isolated
GnRH neurons express low levels of estrogen receptor-
(ER) and/or
ERß primary transcript (12), it is as yet unclear whether this
results in the generation of functional proteins. Similarly, the
effects of adenylyl cyclase inhibition with SQ 22536 were probably not
mediated by inhibition of adenylyl cyclase in GnRH neurons, as
additional treatment with P restored LH surges in these animals; again,
the probable absence of PRs in GnRH neurons makes this scenario
unlikely.
If cAMP represents the manifestation of a daily neural signal for the surge, then are its actions mediated by PR trans-activation, as proposed in our experimental model? Pretreatment of rats with the antiprogestin RU486 resulted in complete blockade of E2-induced LH surges regardless of whether they were temporally advanced by 8-bromo-cAMP. This action of RU486 was very likely a result of central PR antagonism, as similar treatments using this antiprogestin have been shown to decrease GnRH surge secretion on proestrus in rats (13) and to be ineffective in altering pituitary responses to exogenous GnRH in both rats and primates (13, 14). P administration, moreover, effectively restored LH surges in rats treated with the adenylyl cyclase inhibitor SQ 22536 icv. Thus, PR blockade prevents surge release induced by cAMP activation, whereas the effects of cAMP blockade can be reversed by PR activation. These results confirm the obligatory involvement of PRs in the E2-induced GnRH surge, and more importantly, they indicate that PR activation is a necessary signaling event that occurs downstream of cAMP accumulation. These findings are again consistent with the idea that neural signals for the GnRH and LH surges are mediated by cAMP generation, and that these signals trigger GnRH surges by evoking the trans-activation of PRs in the AVPV.
It cannot be determined from these studies whether cAMP may activate PR in a ligand-dependent or a ligand-independent manner. It is possible that liganded PRs may be involved in this signaling event, as some P is produced (15, 16), along with cytochrome P450 (17, 18) and 3ß-hydroxysteroid dehydrogenase (19), in the brain. There is no evidence available, however, to support the idea that signal changes in P occur within neurons in response to neural signals, or that the exceedingly low levels of brain P (15) can activate PRs in a physiological manner. It is also not clear that any residual plasma P levels in OVX rats would provide sufficient concentrations of ligand for binding PR in these animals (20). By contrast, many studies, both in vitro and in vivo, have demonstrated the ability of cAMP to activate PR in the absence of P. cAMP has been shown to trans-activate PR in stably transfected CV-1 and COS-1 (7) cells as well as in enriched gonadotropes from primary pituitary cell culture (6). In vivo studies have demonstrated that DA agonists require unliganded PR to influence lordosis behavior in mice (9). As DA putatively acts only via its membrane-bound G protein-linked receptor, it is logical to assume that any trans-activational effects on PR by DA would be occurring via an increase in the neurotransmitter’s second messenger pathway activity. Other evidence suggests that phosphorylation of dopamine- and cAMP-regulated phosphoprotein (DARPP-32) increases due to mating stimulation, and that this protein may play a role in transducing DA’s effects on PR activation in the VMH and other hypothalamic regions (21). Exactly how increased cAMP may activate unliganded PR remains unclear, although evidence indicates that this trans-activation requires activation of protein kinase A and subsequent phosphorylation of the receptor (22), which then would bind to DNA and increase transcriptional activation of a gene(s) as yet unknown. The availability and activation state of a multitude of transcriptional coactivators and coregulators could also provide a mechanism by which this cross-talk could be mediated, but as many of the characteristics and actions of these factors still remain undefined, further research will be required to determine their roles in this process.
Previous studies have shown that an intact AVPV is required for
generation of LH surges in intact and steroid-primed rodents (23, 24, 25),
and ER activation in this region is critical for surge generation (26);
ER activation also leads to PR messenger RNA and PR protein
accumulation in the AVPV (27). Our studies have additionally provided
evidence that cAMP-induced trans-activation of these PRs in
the AVPV provides a neural signal for the surge. Which
neurotransmitters may convey the daily neural signals to the AVPV,
culminating in cAMP production, PR trans-activation, and
release of GnRH surges? Candidate neurotransmitters include vasoactive
intestinal peptide (28), vasopressin (29), and 
-aminobutyric acid
(30), as neurons expressing these transmitters have been identified
among those located in the SCN and send projections to the AVPV (25, 31). Furthermore, these neurotransmitters have been found to be
produced in the expected rhythmic fashion, and some (vasopressin and
vasoactive intestinal peptide) are capable of activating cognate
receptors that are positively coupled to adenylyl cyclase (32, 33, 34).
It also remains to be determined how PR trans-activation may
lead to GnRH surge generation. The 2- to 3-h latency between
8-bromo-cAMP administration and the stimulation of LH surges is
consistent with the idea that the actions of PR in AVPV neurons are
mediated by regulation of gene transcription. Much information has been
obtained regarding the neurotransmitters produced by subpopulations of
AVPV neurons. Earlier studies have shown that ER-immunoreactive cells
in AVPV receive extensive input from SCN and also send afferents to
GnRH perikarya (35, 36), and the neurotransmitters produced by these
cells include -aminobutyric acid, dynorphin, enkephalin, DA, and
substance P, among others (25). Given these observations, it is
possible that trans-activated PRs in these neurons regulate
the expression of genes encoding either these neurotransmitters or the
enzymes involved in their synthesis. Alternatively, activated PRs may
also regulate the expression of a variety of proteins that regulate
cell excitability or secretion. We have recently demonstrated, for
example, that PR activation mediates increased hypothalamic
neuropeptide Y1 receptor gene expression (37), which, in turn, results
in heightened responsiveness of the GnRH secretory system to
neuropeptide Y stimulation during the initiation of GnRH and LH
surges.
Convergence of two signaling pathways at steroid receptors has been proposed to be involved in other reproductive pathways, most notably the GnRH self-priming response by pituitary gonadotropes (6) and induction of sexual behavior by neurotransmitters in the VMH (38). Our results suggest that a similar mechanism may be required for timing and initiation of GnRH surges. Although these experiments focused primarily on E2-induced PRs and their activation by cAMP, there is evidence that E2 may exert additional actions mediated by E2’s ability to induce expression or activation of other transcriptional regulators. Both E2 and P treatment, for example, have been shown to rapidly phosphorylate cAMP response element-binding protein (CREB) in cells of various neuronal phenotypes in the AVPV (39), suggesting that an increase in phospho-CREB by steroids could play a part in regulating gene expression in this region. The relevance of rapid E2-induced alterations in phospho-CREB, however, has yet to be analyzed in the context of the initiation and timing of GnRH and LH surges; it is possible that E2’s effects on CREB are one of several amplification events that occur downstream from cAMP-induced PR activation, including E2 priming of pituitary gonadotropes (40).
These studies provide evidence for the validity of a basic tenet of an integrative model for GnRH surges; a daily neural signal, comprised of a transient increase in cAMP in AVPV neurons, can evoke activation of E2-induced PRs and thereby stimulate the release of GnRH surges. It remains unknown whether this cross-talk mechanism figures as importantly in the control of GnRH and LH surges in other species, particularly those animals in which these events are not under the strict control of circadian timing signals. The prevalence of this type of integrative mechanism in the control of other neuroendocrine systems also remains to be determined. Cross-talk between membrane-bound and intracellular receptor-mediated signaling pathways has been established for a variety of cellular signaling pathways, such as growth factor stimulation of ER-dependent breast cancers (41). Future studies are likely to reveal that analogous cellular mechanisms mediate the permissive effects of a variety of other hormones on neurophysiological and behavioral processes.
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Acknowledgments |
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Received October 4, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß messenger ribonucleic acids in adult
gonadotropin-releasing hormone neurons. Endocrinology 140:5195–5201
5-4-isomerase
(3ß-HSD), is expressed in rat brain. Brain Res Mol Brain Res 30:287–300[Medline]
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