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Institut National de la Santé et de la Recherche Médicale U422, Institut Fédératif de Recherches 22, Unité de Neuroendocrinologie et Physiopathologie Neuronale, 59045 Lille Cedex, France
Address all correspondence and requests for reprints to: Claude Knauf, Institut National de la Santé et de la Recherche Médicale U422, Institut Fédératif de Recherches 22, Unité de Neuroendocrinologie et Physiopathologie Neuronale, 59045 Lille Cedex France. E-mail: knauf{at}lille.inserm.fr
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Abstract |
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Introduction |
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Among a wide range of physiological and behavioral processes, NO has been implicated in the regulation of prehypophysiotropic secretions at the median eminence (ME) level (13). More precisely, NO may participate in the regulation of the GnRH neurons by stimulating GnRH release from its neuroendocrine terminals (14, 15, 16, 17, 18), located in the external zone of the median eminence. In addition, in a recent work, we demonstrated that ME fragments spontaneously release NO (19). This NO generation is likely to be pulsatile and cyclic across the estrous cycle. Moreover, the highest NO concentrations are measured on proestrus and correlated to GnRH release
At that time, the origin of NO implicated in the GnRH secretion remains to be clearly determined. While nNOS has been detected in cell processes in the internal zone generally far away from GnRH nerve endings, eNOS immunoreactivity was restricted to the vascular tissue (20), very close to the neuroendocrine nerve terminals. Thus, endothelium might be the major source of NO implicated in the GnRH release in the ME, although some authors consider that NO has rather a neuronal origin (14, 21). The endothelial hypothesis is consistent with the facts that 1) rat ME fragments stimulated with exogenous substances, such as E2 or morphine, are able to release NO from vascular endothelium (17, 18); 2) addition of L-NIO, a preferential eNOS inhibitor when used at certain concentrations, induces a decrease in the spontaneous NO/GnRH release from rat ME (18, 19); and 3) intracerebroventricular (icv) injection of eNOS antisense is more efficacious than nNOS antisense in inhibiting the E2/progesterone-induced GnRH/LH surge in ovariectomized female rats (22).
The fact that NO might originate from endothelium is relevant because it points out an important role of endothelial cells in neuroendocrine regulations that constitute a new concept that we have advanced in our last results (17, 18, 19). However, further data are needed to definitely consider this concept as accepted. In this perspective, the aim of this work is essentially to evaluate the variations of eNOS expression in the ME during estrous cycle using RT-PCR and Western Blot experiments. As concentrations of circulating gonadal steroids are known to vary across the estrous cycle, the part played by E2 and progesterone in controlling eNOS expression was also investigated in the ME of steroids-treated ovariectomized (ovx) female rat. In regard to the possible specific inhibitory action of cav-1 on eNOS activity, we also focus our attention on the possible existence of cav-1 protein in the ME. As a consequence, levels of the inhibitory cav-1 protein were studied in the ME of cycling or steroids-treated ovx female rats. This work supplies evidence that eNOS expression in the ME is steroid dependent and higher the day of proestrus, reinforcing the idea that NO of endothelial origin has an important role on GnRH release. Moreover, the inhibitory cav-1 protein seems to be an important modulator of eNOS activity in the ME. These results give additional proof for an essential role of vascular endothelium in controlling NO/GnRH release.
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Materials and Methods |
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Female cycling rats
Vaginal lavage of female rats was examined daily, and only rats
that exhibited at least two consecutive 4-d estrous cycles were used
for the present experiments. Female rats were killed by decapitation at
1400 h on diestrus II (DiII); at 0800 h, 1600 h,
1800 h on proestrus (Pro); and at 0800 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 (23).
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. For each stage of the estrous
cycle, 9 animals were killed to obtain sufficient quantities of total
RNA (4 animals) for RT-PCR and proteins (5 animals) for Western
blotting.
Ovariectomized rats
Twenty-seven animals were bilaterally ovariectomized (ovx) under
ether anesthesia. The animals were divided in three groups: 9 animals
were killed at 1400 h on d 17 without receiving any treatment; 9
animals received a single sc injection of E2 (30 µg/rat) at 1000
h on day 15 and were killed at 1400 h on day 17; and 9 animals
received sc 30 µg of E2 at 1000 h on day 15 and 2 mg of
progesterone (P) at 1000 h on day 17 and were killed the same day
at 1400 h. In these experimental conditions, E2 + P treatment to
ovx rats induces the LH surge at 1400 h (16).
After dissection, the ME fragments were stocked at -80 C.
RT-PCR of eNOS and 28S
Total RNA from ME (4 ME) was extracted using RNeasy kit
(QIAGEN, Les Ulis, France) according to the
manufacturer’s instructions. A quantity of 0.5 µg of the total RNA
was reverse transcribed using the Superscript One Step RT-PCR system
(Life Technologies, Inc., Cergy Pontoise, France).
eNOS-specific primers were similar to those used by Keilhoff et
al. (24). The sequence of the primers used for PCR
were sense 5'-GAG AAT TCC ACC TCA CTG TAG CTG TAG CTG TGC TGG CA-3';
and antisense 5'-TCG AAT TCC CAG GGC ACT GCG CCC CGC AAC TG-3', and
allowed the amplification of a 740-bp product. As internal control, the
28S cDNA was amplified with the specific primers (sense: 5'-GCA GGG CGA
AGC AGA AGG AAA CT-3'; antisense: 5'-TGA GAT CGT TTC GGC CCC AA-3'),
which should amplify a 229 bp product. RT was performed at 55 C for 30
min in a Biometra T Gradient cycler and followed by 30 cycles (eNOS) or
14 cycles (28S) consisting of denaturation at 94 C for 1 min, annealing
for 1 min at 63 C (eNOS) or 60.9 C (28S), and DNA extension at 72 C for
2 min 30. Under these experimental conditions, the linearity of the
amplification was observed up to 40 cycles for eNOS and 16 cycles for
28S. PCR products were subjected to electrophoresis on a 2% agarose
gel containing ethidium bromure. The estimation of the band size was
performed using the 100-bp DNA ladder marker (Life Technologies, Inc.). The identity of the amplified eNOS product was confirmed
by restriction enzyme digestion with AluI, AvaI,
and EcoRI.
Detection of eNOS and cav1 proteins by immunoblotting
Tissues fragments (5 ME) of the same stages were recovered
in ice cold lysis buffer (50 mM Tris, pH 7.4, 1% NP-40,
1% Triton, 150 mM NaCl, 1 mM EDTA) with
protease inhibitors (complete, mini, Roche Molecular Biochemicals, Neuilly, France), sonicated and rocked
overnight at 4 C. The total cell lysate is recovered in supernatant
after centrifugation at 12,000 x g at 4 C for 20 min.
Protein concentration was determined using the Protein Assay Reagent
BCA kit (Pierce Chemical Co., Madison, WI).
Equal amounts of the solubilized proteins from ME were separated in SDS-polyacrylamide gel (ratio acrylamide/bis acrylamide 37.5/2) and electrotransferred to a nitrocellulose membrane. The membranes were blocked for 30 min in TNT buffer (Tris-buffered saline, pH 8, with 0.05% Tween) containing 5% milk. Then, membranes were incubated for 1 h 30 at room temperature with polyclonal antibody against eNOS (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; dilution: 1/500), cav-1 (Santa Cruz Biotechnology, Inc., dilution: 1/1000) and neuronal specific enolase (NSE, Tebu, dilution: 1/50000) as internal control. Antibodies for eNOS, cav-1, and NSE were added successively on to the same membrane. After washing three times with TNT buffer, membranes were incubated with horseradish peroxidase conjugated antirabbit IgG antibody (Sigma, Saint Quentin, France). Immunoreactivity was detected using an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, Orsay, France).
Results analysis
Concerning RT-PCR studies, bands intensity was determined by the
use of the Kodak Digital Science program. Blots
quantification was performed by Image Master 1D program (Amersham Pharmacia Biotech).
All experiments were repeated a least of 4 times.
Statistical analysis
All data are presented as the mean ± SEM. Data
were analyzed by one-way ANOVA. A Dunnett test was performed to compare
the data shown on figures. The level of significance was set at
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). NSE served as internal control (Fig. 1B). eNOS protein levels during the estrous cycle were determined by
normalization of eNOS densitometric values to NSE ones (Fig. 1C). eNOS
protein levels significantly increased on 0800 h and 1600 h
proestrus, compared with 1400 h diestrus II, then returned to
basal value on 1800 h proestrus and 0800 h estrus
(P < 0.05).
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).
Densitometric values of eNOS were normalized with respect to those of
28S (Fig. 2B). Our results demonstrate a significant increase in mRNA
eNOS levels on proestrus 0800 h and 1600 h compared with
diestrus II 1400 h (P < 0.05). No significant
variation was observed on proestrus 1800 h and estrus 0800 h.
These results suggested that eNOS mRNA level is transiently augmented
on proestrus day.
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Variations of eNOS protein levels in the rat ME in experimental
conditions. We analyzed eNOS protein levels in the ME from these
animals by Western blot (Fig. 3A). NSE
was also analyzed as internal control (Fig. 3B), and eNOS level has
been normalized with respect to NSE (Fig. 3C). The eNOS protein levels
significantly increased in the ovx + E2 rat ME and in ovx + E2 + P rat
ME (P < 0.05).
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). As shown in Fig. 4B, treatment with
E2 of ovx rats caused a very important elevation eNOS mRNA in the ME
(P < 0.05). The additional supplementation with P led
to a decrease of eNOS mRNA concentrations to nadir levels,
i.e. similar to the one observed in ovx animals (Fig. 4B).
These results suggested that E2 exerts a stimulatory effect on eNOS
transcript that seems to be blocked by progesterone.
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). High levels of cav-1 protein were
detected on diestrus II 1400 h and proestrus 0800 h. Levels
of cav-1 on proestrus 1800 h and estrus 0800 h were
intermediary (Fig. 5B). Western blot analysis of cav-1 in ME show that
E2 and E2 + P treatments in ovx animals result in a decrease of cav-1
protein levels (Fig. 6). Consequently,
steroids may have an opposite regulatory role on eNOS and cav-1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The part played by NO in controlling GnRH release into the fenestrated pituitary portal blood vessels of the median eminence is now evident. Inhibition of the NO pathway by icv administration of NOS inhibitors or antisense provokes a lack of the GnRH/LH secretory activity (14, 22). At the ME level, eNOS immunoreactivity was observed at the external zone, very close to the GnRH terminals, as opposed to nNOS immunoreactive cells located in the internal zone of the ME (20, 25). In this regard, a new idea has emerged suggesting that NO might rather have an endothelial origin and has lead to the concept that vascular endothelium might have a role in neuroendocrine controls (25). Indeed, although several authors have demonstrated the importance of nNOS in controlling GnRH cell bodies activity at the preoptic area and in the ME (14, 21, 26), the majority of NO measured at the ME level might be endothelial in origin. Using amperometric probes, Prevot et al. (17, 18) show that rat ME fragments, stimulated with exogenous substances, release NO and give arguments for an endothelial origin. The same authors demonstrate that this NO stimulates GnRH release. The endothelial origin of NO secreted from ME fragments is also consistent with the work of Aguan et al. (22), showing that icv administration of eNOS antisense is more efficacious than nNOS antisense in suppressing the E2/progesterone-induced GnRH/LH surge in ovx female rats. The role of eNOS in reproductive functions is reinforced by the observation that eNOS KO female mice present dysfunction in cyclicity, ovulation rate, ovarian morphology, and steroidogenesis (27). Nevertheless, in spite of the disorder, eNOS KO female mice are still fertile suggesting that the loss of the function of the protein derived from the disrupted gene is compensated by other mechanisms.
Recently, we provided compelling evidence demonstrating the importance of NO in controlling GnRH release during the estrous cycle (19). Indeed, spontaneous NO release from ME fragments from cycling female appeared to be pulsatile and cyclic, in parallel to GnRH release. NO significantly increased from 0800 h proestrus to 1600 h proestrus. In this previous study, the NO pulsatility recorded at the ME level was significantly inhibited by L-NIO at 5*10-7 M, a preferential eNOS inhibitor at this concentration (28). In the present data, by evaluating the level of eNOS expression in rat ME across the estrous cycle, we show that the increase of NO release on proestrus is concomitant to an increase in eNOS synthesis. This observation reinforces the idea that endothelium is implicated in the GnRH release during the estrous cycle.
The following point to answer now is: what is able to stimulate eNOS during the estrous cycle? Activity and expression of eNOS are regulated by various molecules (29), including E2. The fact that eNOS variations are observed during the estrous cycle naturally suggests that steroids are implicated in this phenomenon. In addition, a previous work from our laboratory has also reported that E2 was able to induce very rapidly a secretion of NO, suggesting the implication of a membrane receptor for E2 (18). Besides this rapid effect, it has also been demonstrated the existence of long-term effect of E2 on eNOS mRNA (30) and protein synthesis in endothelial cells (31). Altogether, these results strongly lead to think that the increase in eNOS levels are linked to steroid variations. More precisely, it seems that E2 is the main gonadal steroid responsible for this increase: 1) treatment of ovx with E2 increases NO secretion (18, 19) and stimulates eNOS expression in the present work while; and 2) the subsequent treatment with P of ovx + E2 animals decreases NO secretion (19) and suppresses the stimulatory effect of E2 on eNOS mRNA expression. However, it can be noted that, in these experimental conditions, levels of eNOS protein remain high after P supplementation, suggesting that P has an inhibitory effect on eNOS mRNA expression but not on eNOS mRNA translation. Conversely in physiological conditions, i.e. during the estrous cycle, both eNOS protein and eNOS mRNA levels drop between proestrus 1600 h and proestrus 1800 h and remain low on estrus. At the moment, little is known about P influences on NOS transcription. It had been demonstrated that treatment with P caused a time- and dose-dependant inhibition of NO production by macrophage cell lines (RAW 264.7, J774) by reduction of iNOS mRNA (32, 33). We surmise that after the E2-stimulatory effect on eNOS, P may inhibit eNOS gene expression, to come back to basal value of eNOS mRNA observed from 1800 h proestrus to diestrus II. Important eNOS protein concentration observed in Ovx + E2 + P animals are probably due to a high previous synthesis of the enzyme, in response to E2 in this experimental condition.
As stated earlier, eNOS is regulated by a transmembrane protein, cav-1. Indeed, in vascular endothelial cells, eNOS is targeted to plasmalemmal caveolae (34), where it interacts with cav-1. The interaction of eNOS with cav-1 occurs by direct protein-protein interaction that involves 20 amino acids region within the cav sequence (35, 36), termed the "caveolin scaffolding domain" (37). Coupled to cav-1, eNOS enzyme is tonically inhibited. The dissociation of cav-1 from eNOS directly activates the enzyme, which translocates to intracellular sites (34). In the present study, the increase in eNOS protein expression and the decrease of cav-1 protein expression at the ME level on the afternoon of proestrus demonstrate that the high eNOS activity recorded was due to an inverse mode of regulation of two interactive molecules. The combination of increased eNOS and decreased cav-1 expression under the influence of E2 has been previously observed in pial arteriolar by Pelligrino et al. (38). It has been shown that E2 stimulates eNOS expression and activity, through down-regulation of cav-1. However, the ability of E2 to modify eNOS activity/expression via controlling cav-1 expression is only one possibility to explain the variations of eNOS activity on proestrus. Indeed, in addition to myristoylation and palmitoylation that permitted the interactions with cav-1, eNOS can be modified by phosphorylation (39). The phosphorylation of eNOS is thought to regulate enzyme activity in a calcium-independent fashion (40, 41), but the signaling pathway implicated is not yet known. For example, several authors have focused their attention on the Akt kinase pathway, which can directly phosphorylate eNOS and activate the enzyme (42, 43). This way may be further explored in our amperometric model, which did not take into account the rapid and calcium-independent mechanism involved in eNOS activation.
In summary, our results demonstrate that eNOS/cav-1 levels are modified during the estrous cycle in an inverse profile, probably in response to variable plasmatic concentrations of E2. The high levels of NO that are observed concomitantly to the GnRH surge on the afternoon of proestrus seem consequently to be the results of an increase in eNOS synthesis coupling with a decrease in the concentration of the inhibitory protein cav-1.
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Acknowledgments |
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Footnotes |
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Abbreviations: CaM, Calmodulin; eNOS, endothelial nitric oxide synthase; icv, intracerebroventricular; iNOS, inducible NOS II; ME, median eminence; nNOS, neuronal-type nitric oxide synthase I; NO, nitric oxide; NOS, nitric oxide synthase; NSE, neuronal specific enolase.
Received April 16, 2001.
Accepted for publication June 28, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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