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Institut National de la Santé et de la Recherche Médicale U422 (C.K., V.P., G.M., J.-C.B., D.C.), IFR22, Unité de Neuroendocrinologie et Physiopathologie Neuronale, 59045 Lille Cedex, France; Neuroscience Research Institute (G.B.S.), State University of New York, Old Westbury, New York 11568; and the Division of Psychiatry (G.B.S.), Harvard Medical School, Brigham and Women’s Hospital, Boston, Massachusetts 02115
Address all correspondence and requests for reprints to: Vincent Prevot, Oregon Regional Primate Research Center/Oregon health Science University, 505 Northwest 185th Avenue, Beaverton, Oregon 97006. E-mail: vincentp{at}ohsu.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NO has been implicated in reproductive functions and behaviors, including ovulation, oocyte meiotic maturation (11), estradiol (E2)-synthesis (12), lordosis, and penile erection (13). NO also participates in the control of GnRH/LH release by modulating GnRH neurons activity at the hypothalamus (14, 15, 16, 17). At the preoptic area, the close anatomical interrelation between nNOS-immunoreactive cells and the GnRH perikarya (18) supports the speculation of NO controlling GnRH cell body activity. At the median eminence (ME), projection site of the GnRH neurons, NO stimulates GnRH release from the GnRH neuroendocrine terminals located within the ME external zone (15). The fact that intracerebroventricular injections of NOS inhibitors or antisense oligonucleotides inhibit the GnRH/LH secretory activity (15, 19) suggests that NO plays a major role in the control of the GnRH neuroendocrine axis and thus in reproductive functions (14).
Ex vivo studies on ME or mediobasal hypothalamus fragments demonstrated that NO exerts at least part of its stimulatory effect on GnRH release by acting directly on GnRH nerve terminals (15, 16, 17). NO might target either the guanylyl cyclase (16, 20, 21) or the cyclooxygenase (22) within the GnRH nerve terminals. These enzymes generate cyclic GMP (cGMP) and PG E2 formation. Induction by cGMP of a cationic conductance (23) stimulates a depolarization from neuroendocrine terminals that leads to GnRH release (24), whereas PG E2 enhances the mobilization of Ca2+ from intracellular stores (25) and cAMP formation (26) that induces the exocytosis of GnRH secretory granules (22). Thus, known NO signaling cascades can account for the reported pharmacological actions of this signaling molecule.
Within this last decade, many studies have attempted to identify the endogenous neuroendocrine pathways involved in the control of NO-stimulated GnRH release. However, little is known about the origin of NO locally produced at the ME, and nothing is known about its pattern of secretion. Given that iNOS is undetectable in the hypothalamus under normal conditions, we surmise that NO is synthesized in the ME by a constitutive NOS (20, 27) because, by definition, it is always present. In this regard, nNOS is detected in fibers located in the internal zone of the ME projecting to the neuropituitary (18), and eNOS has been located in endothelial cells situated very close to the neuroendocrine GnRH terminals of the ME external zone (28). In previous reports, we demonstrated that at the ME, the NO of vascular origin is able to modulate hypothalamic neurosecretions, i.e. GnRH release (20, 27). The aim of the present study is to determine whether, under physiological and experimental circumstances, we are able to detect NO secretion from ME fragments without the need for exogenous stimulatory substances, by using the amperometric method for NO detection. This study provides compelling evidence for the importance of the vascular endothelium in the control of GnRH release.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Exp 1: analysis of spontaneous NO release from ME fragments
throughout the rat estrous cycle
Vaginal lavage of female rats was examined daily, and only rats
that exhibited at least 2 consecutive 4-day estrous cycles were used
for the present experiment. Female rats were killed by decapitation at
0800 h and 1400 h on diestrus II (DiII); at 0800 h,
1400 h, 1600 h, 1700 h, and 1800 h on proestrus
(Pro); and at 800 h and 1400 h on estrus (E). After rapid
removal of the brain, the ME and associated vascular tissues were
dissected under a binocular magnifying glass by cutting with Wecker’s
scissors (Moria, France) the floor of the brain within the following
limits: posterior border of the optic chiasma and the anterior border
of the mamillary bodies, as described before (20).
Following this dissection procedure, pieces containing ME were obtained
with very little arcuate nucleus fragments. The total dissection time
was less than 3 min from decapitation. After dissection, the ME
fragments were washed twice in Krebs-Ringer bicarbonate/glucose buffer
(pH 7.4) containing 23 µM bacitracin (Sigma,
St. Louis, MO) in an atmosphere of 95% O2-5%
CO2 and then immersed in Eppendorf
tubes containing 800 µl of the same medium. Each tube contained one
ME fragment. After a 30-min recovery period, the medium was changed,
and then the spontaneous NO release was measured at 35 C for 4 periods
of 30 min by using a NO-specific amperometric probe (World Precision
Instruments, Sarasota, FL) as previously described (20, 27). At the end of each 30-min incubation-period, 600 µl of
medium was removed and replaced with 600 µl of fresh Krebs-Ringer
bicarbonate/glucose buffer. EDTA
(10-2 M final)
was added to the removed medium. Because the amperometric probe
requires at least a 5-min recovery period after each change of medium,
we were unable to shorten the intervals of sample collection without
negatively affecting the real-time NO release data acquisition. Each
sample was stored at -20 C before analysis of the GnRH levels in the
medium. The tip diameter of the amperometric probe (25 µm) used to
monitor NO secretion permitted the use of a micromanipulator
(Carl Zeiss-Eppendorf, Hambourg, Germany) to
position the sensor 5 mm above the tissue surface. Calibration of the
electrochemical sensor was performed by the use of different
concentrations of a nitrosothiol donor S-nitriso-N-acetyl-D,
L-penicillamine (Sigma), as
previously described in detail (29). The concentration of
NO gas in solution was measured in real-time with data acquisition (DUO
18, World Precision Instruments) at a sampling rate of 6/sec
(20). The computer-interfaced DUO-18 software (World
Precision Instruments) performed data acquisition. The experimental
values were then transferred to Sigma-Plot and
Sigma-Stat (Jandel, San Rafael, CA) for graphic
representation and evaluation.
Exp 2: effect of an eNOS selective inhibitor on NO/GnRH release
from Pro 1600 ME fragments
To assess whether the major source of NO secreted at the ME is,
as suggested by our previous studies (20, 27), mainly
endothelial in origin, rather than neuronal, we treated Pro 1600 ME
fragments with
L-N5-(1-iminoethyl)ornithine
(L-NIO) at 5 x
10-7
M. At this concentration,
L-NIO selectively inhibits the eNOS activity
(30) and suppresses the NO- induced GnRH release from
male ME fragments in the rat (20). The ME fragments were
processed as described above. The L-NIO was added
after the washes throughout the whole incubation period.
Exp 3: effect of ovarian steroids on ME NO release
Twelve animals were bilaterally ovariectomized (OVX) under ether
anesthesia. The animals were divided in three groups: four animals were
killed at 1400 h on the 17th day after OVX without receiving any
treatment; four animals received a single sc injection of
E2 benzoate (E2B, 30
µg/rat) at 1000 h on day 15 and were killed at 1400 h on
day 17; and four animals received sc 30 µg E2B
at 1000 h on day 15 and 2 mg progesterone (P) at 1000 h on
day 17 and were killed at 1400 h the same day. Amperometric
measures of NO release were conducted as described above.
GnRH RIA
GnRH concentrations were measured in the medium for each 30-min
incubation period for each animal of any experimental design, in
duplicate by RIA, according to Nett and Adams’ method
(31), with minor modifications. Monoiodinated GnRH was
isolated using a QAE Sephadex column. Antiserum raised to GnRH-HSA was
produced in rabbit; and after absorption by HSA, it was used to a final
dilution of 1:140,000. The sensitivity for GnRH was 1.2 pg/tube, and
intraassay variability was 3.4%. The GnRH antibody was a gift from Dr.
Tramu of the Centre National de la Recherche Scientifique URA
339, Université Bordeaux I (Talence, France).
Statistical analysis
All experiments were repeated a minimum of four times,
i.e. a minimum of four animals was run for each experimental
condition. Within the 2 h-long amperometric record obtained for each
ME, the maximal amplitude of NO secretion for each one of the four
30-min incubation periods was taken into account and averaged.
Similarly, for each experiment, the GnRH levels were determined by
averaging the GnRH concentration of each 30-min incubation period
sample. The mean NO/GnRH release for each time point of the estrous
cycle or each experimental condition was reported on the different
graphs (see
Figs. 2–4
). To facilitate the comparison of NO
release during the different stages of the estrous cycle, the area
under the curve (AUC) was calculated for each 30-min incubation period.
All data are presented as the mean ± SEM.
Data were analyzed by one-way ANOVA. Student’s-Newman-Keuls multiple
comparison (see Figs. 1 and 4) was used post hoc to find
significant differences between groups. A Dunnett test was performed to
compare the data shown on Table 1. A
Student’s t test was used to compare the two different
groups (see Fig. 3). To determine whether the GnRH levels monitored
throughout the estrous cycle were linked to the NO levels, a Pearson
product Moment Correlation was performed. The level of significance was
set at P < 0.05.
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Results |
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). NO efflux was pulsatile (Table 1),
and its amplitude varied significantly across the estrous cycle (Figs. 1 and 2A). NO secretion was lowest on
diestrus II. It increased significantly on the morning of Pro
(P < 0.05) and reached a maximum on Pro 1600 h.
At Pro 1800 h, the NO levels were significantly decreased
(P < 0.05), and nadir levels were maintained through E
(Fig. 2A). Noticeably, as illustrated in Fig. 1, the NO peak
concentrations reached 30–50 nM on Pro, whereas
they rarely exceeded 20 nM on diestrus and E.
Cyclic NO and GnRH secretions during the estrous cycle are
coupled
The collection of the incubation medium every 30 min
demonstrated that the GnRH secretion from the neuroendocrine terminals
located in the external zone of the ME followed a release profile
identical to the one observed for NO secretion (Fig. 2B),
i.e. the GnRH secretion was basal on diestrus II, increased
on Pro, and reached a maximum on Pro 1600 h. On E, the GnRH levels
were significantly lower, when compared with those on Pro 1600 h,
but remained higher than on diestrus 0800 h. The Pearson product
moment correlation test revealed that across the estrous cycle, the
GnRH secreted levels were positively and strongly correlated to NO
secretion (GnRH secreted vs. AUC: r = 0.86,
P < 0.003, n = 9).
Selective inhibition of eNOS activity potently inhibits NO/GnRH
release on Pro 1600 h
To test the hypothesis that the GnRH secretion from ME
fragments observed on Pro was actually linked to NO secretion, we used
L-NIO, a potent NOS inhibitor, to inhibit the spontaneous
NOS activity in the ME. Additionally, we determined which NOS was
involved in the spontaneous secretion of NO at the ME by using
L-NIO at 5 x
10-7 M, a
concentration that selectively inhibits eNOS (20, 30). The
addition of 5 x 10-7
M L-NIO into the survival medium of Pro 16
h ME fragments significantly inhibited (P < 0.01) both
the strong NO secretory activity and GnRH release (Fig. 3). As found in Fig. 3, 5 x
10-7
M L-NIO did not totally
suppress the ME NO release. In part, this may arise from the fact that
5 x 10-7
M L-NIO corresponds to the
concentration required to produce 50% inhibition
(IC50) of eNOS (30) and is 10 times
lower to the IC50 for nNOS (32),
suggesting a limited level of nNOS activity and/or that a low level of
eNOS activity remains in the ME fragments after
L-NIO treatment (see 53).
E2 treatment stimulates ME-fragment NO release in OVX
rats (correlation with GnRH release)
To assess whether estrogen was involved in the increase of NO
secretory activity between diestrus 1400 h and Pro 0800 h and
to determine the role that progesterone may play in the regulation of
GnRH/NO activity on Pro, OVX rats were either untreated or treated with
E2B, or E2B followed by
progesterone, and NO/GnRH release was monitored from ME fragments
obtained from these animals. Two weeks after OVX, both the NO and GnRH
effluxes from ME fragments were low and comparable with that monitored
on diestrus II (Fig. 4). Forty-eight-hour
treatment of OVX animals with E2B resulted in a
significant increase (P < 0.05) in NO secretion that
was coupled with a significant increase in GnRH release
(P < 0.05). In
E2B+progesterone-treated animals, the NO levels
were higher than in untreated OVX rats, but not significantly
different, whereas GnRH secreted levels were significantly higher
(P < 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Within the last decade, NO has been shown to be a key neurotransmitter involved in the control of GnRH/LH secretion in both males and females. NO release controls basal LH secretion in males (15) and mediates the ovarian steroid-induced LH surge in females (19, 33). Studies carried out in vivo (21) suggest that the NO secreted at the hypothalamic preoptic area by nNOS neurons surrounding GnRH cell bodies (18) may be involved in the control of GnRH/LH secretion. On the other hand, our results demonstrate that the NO secreted at the ME plays a crucial role in the control of GnRH release from the neuroendocrine terminals during the female rat estrous cycle. This ME NO secretion may contribute to the synchronization of GnRH release from the anatomically scattered GnRH nerve terminals located in the external zone of the ME, as suggested by in vitro studies carried out in cell lines (34). In the immortalized GT1–1 cell line, secreting GnRH in vitro (35) and expressing both nNOS (33, 36) and eNOS (36), the inhibition of these NOS activities by NOS blockers and NO scavengers abolishes the pulsatile GnRH secretion. The authors concluded that NO was the unique neurotransmitter that was necessary to synchronize GnRH neuron activity leading to the establishment of a synchronized pulsatile GnRH secretion (33). However, in the hypothalamus, the GnRH neurons have been shown, thus far, to be devoid of NOS immunoreactivity (18) and, further supporting this observation, they do not exhibit NADPH diaphorase activity (V. Prevot, personal communication) but do secrete their neurohormone in a pulsatile manner into the portal blood capillaries (37, 38). Additionally, NO-donors have been shown, by push-pull perfusion, to directly affect GnRH release (39). Taken together, we surmise that a hypothalamic source of NO, exogenous to GnRH neurons, might control their secretion.
As stated earlier, preoptic area nNOS neurons may play an important role in the control of GnRH cell body activity and thus GnRH/LH secretion; but because they are only closely associated with GnRH neurons in the rostral preoptic area (18), they are unlikely to be the synchronizing link between the scattered GnRH neurons. nNOS neurons may rather relay stimulatory or inhibitory inputs to GnRH neurons, as is the case for ß-endorphinergic inputs (40, 41). Intriguingly, mediobasal hypothalamus or ME fragments are able to secrete GnRH in a pulsatile manner (42, 43), demonstrating that the GnRH cell bodies are not necessary for pulsatile GnRH release, suggesting that a mechanism of GnRH pulsatility is located in the mediobasal hypothalamus. Our previous studies demonstrated that NO is produced locally at the ME and that its major source is endothelial in origin (20, 27). At that time, we surmised that this endothelial NO might play a crucial role in the synchronization of GnRH release from the nerve terminals spread across the 2.5-mm-long ME (28). In the present study, the strong inhibition of the potent NO/GnRH release on 1600 h Pro by L-NIO, at a concentration that selectively inhibits endothelial NO release at the ME (20), demonstrates that the spontaneous release of NO from ME fragments that participate in the modulation of GnRH release across the estrous cycle is endothelial in origin. Another finding of interest is that the spontaneous NO release monitored across the estrous cycle seems to be pulsatile in female rats. The mean (±SEM) frequency of 1 pulse of NO secretion every 32 ± 1 min (n = 36) in females is strikingly similar to the frequency of pulsatile GnRH secretion from explants of sexually-mature male rats (42). Thus, the findings of the present study strongly suggest that the ME endothelial NO is the synchronizing link to anatomically scattered GnRH neurons and that this synchronizing mechanism takes place at the ME, where it regulates GnRH release directly from the neuroendocrine terminals.
Our results also suggest that the increased endothelial ME NO release on Pro may account for the increase of both GnRH basal release and GnRH pulse amplitude (44) on that day of the estrous cycle. Sarkar and Minami (44) showed in vivo that, as we observe for ME NO release ex vivo, the GnRH basal release and the GnRH pulse amplitude were significantly increased between diestrus and the morning of Pro and that these parameters were maximal the afternoon of Pro, whereas the GnRH interpulse interval did not change. This interplay between endothelial ME NO and GnRH release in the pituitary portal blood is strengthened by the fact that central administration of eNOS antisense oligonucleotides suppresses the steroid-induced LH surge in OVX rats (19).
Estrogen is known to exert a powerful stimulatory influence on GnRH secretion into the portal blood capillaries from the nerve terminals in the ME, to initiate the LH surge (45). Our results strongly support the fact that estrogen could exert part of its stimulatory effect on the GnRH neuroendocrine system by acting directly on the ME. Indeed, the significant increase in ME NO/GnRH secretion in estrogen-treated OVX rats, compared with OVX untreated females, suggests that the dynamic changes in NO/GnRH release observed on the morning of Pro is coupled to the dramatic increase in estrogen plasma levels that occurs between diestrus II and Pro (46). This is in agreement with the findings that estrogen, in addition to directly stimulating eNOS activity at the ME (20), stimulates both eNOS messenger RNA (mRNA) (47) and protein expression (48) in endothelial cells. Surprisingly, progesterone treatment of estrogen-primed OVX rats did not increase NO release significantly, compared with untreated OVX females, suggesting that progesterone is not the major factor regulating the interrelation between NO secretion and GnRH release. However, we cannot rule out the involvement of the progesterone receptor in increased NO/GnRH secretory activity observed on Pro, because the progesterone receptor is a key component mediating the stimulatory effect of estrogen on GnRH, i.e. animals lacking the progesterone receptor fail to show E2-induced LH and FSH surges (49), and progesterone receptor antagonists block the robust GnRH/LH surge induced by estrogen in wild-type animals (50, 51).
The mechanism leading to the pulsatile secretion of NO from the ME endothelium remains to be determined. However, there is some information concerning pulsatile endothelial NO release. In this regard, pulsatile pressure occurring in human blood vessels can release eNOS-derived NO (52). Furthermore, endothelial basal NO from blood vessels and other tissues seems to exhibit a basal pulsatile release pattern, even when observed in vitro (53). The mechanism of this pulsatile NO secretion from endothelial cells seems to occur via mechanotransduction of the eNOS present in the calveolae of the plasma membrane with calveolin, which is an inhibitory protein for eNOS (54). Dissociation of eNOS from calveolin allows its coupling with calmodulin and thus stimulates its activity. Alternative translocation of eNOS from the cell membrane to intracellular sites, uncoupling the enzyme from its activators, and thereby attenuating the formation and release of NO, could also account for NO pulsatile release. Interestingly, these translocation mechanisms may be modulated by estrogen (55). Therefore, we surmise that, at the ME, other factors (neuronal and/or glial in origin) may also participate in the control of these modulatory mechanisms generating NO pulsatile release. Interestingly, the brain endothelial cells express the receptors for two key factors controlling GnRH release at the ME (17, 32, 56, 57, 58), neuropeptide Y (NPY) and glutamate (59, 60, 61). Both N-methyl D-aspartate (NMDA) and NPY have been shown to activate NO pathway in endothelial cells (62, 63). Reinforcing a potential role for NPY in the regulation of ME, endothelial NO release is the demonstration of the expression of the NPY Y1 receptor within the portal blood vasculature (64), together with the finding that estrogen can up-regulate GnRH response to NPY on Pro, through stimulation of Y1 gene expression (65).
In summary, our data demonstrate that the spontaneous NO release at the ME, which may modulate both pulsatile and cyclic GnRH release, provides a unique regulatory mechanism for the GnRH neuroendocrine axis, by which it could control primary events such as puberty, gametogenesis, and ovulation. In addition, our results both confirm and strengthen a role for the vascular endothelium in the control of neurosecretions at the ME.
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Footnotes |
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Received September 5, 2000.
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