help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Smith, M. J.
Right arrow Articles by Wise, P. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smith, M. J.
Right arrow Articles by Wise, P. M.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Endocrinology Vol. 142, No. 7 3006-3013
Copyright © 2001 by The Endocrine Society

ARTICLES

Neurotensin Gene Expression Increases during Proestrus in the Rostral Medial Preoptic Nucleus: Potential for Direct Communication with Gonadotropin-Releasing Hormone Neurons1

Matthew J. Smith and Phyllis M. Wise

Department of Physiology, College of Medicine, University of Kentucky, Lexington, Kentucky 40536

Address all correspondence and requests for reprints to: Phyllis M. Wise, Ph.D., Department of Physiology, University of Kentucky College of Medicine, 800 Rose Street, Lexington, Kentucky 40536-0298. E-mail: pmwise1{at}pop.uky.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neurotensin (NT)-containing neurons in the rostral portion of the medial preoptic nucleus (rMPN) of the brain may play a key role in regulating the pattern of secretion of GnRH, thereby influencing the reproductive cycle in females. The major goals of this study were to determine whether NT messenger RNA (mRNA) levels in the rMPN exhibit a unique pattern of expression in temporal association with the preovulatory LH surge and to assess whether NT neurons may communicate directly with GnRH neurons. We analyzed NT gene expression in rats using in situ hybridization over the day of proestrus and compared this with diestrous day 1. We also determined whether the high-affinity NT receptor (NT1) is expressed in GnRH neurons using dual-label in situ hybridization and whether this expression varies over the estrous cycle. We found that NT mRNA levels in the rMPN increase significantly on the day of proestrus, rising before the LH surge. No such change was detected on diestrous day 1, when the LH surge does not occur. Furthermore, we observed that a significant number of GnRH neurons coexpress NT1 mRNA and that the number of GnRH neurons expressing NT1 mRNA peaks on proestrus. Together with previous findings, our results suggest that increased expression of NT in the rMPN may directly stimulate GnRH neurons on proestrus, contributing to the LH surge. In addition, our results suggest that responsiveness of GnRH neurons to NT stimulation is enhanced on proestrus due to increased expression of NT receptors within GnRH neurons.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CYCLIC SYNTHESIS AND secretion of GnRH is absolutely essential to maintain normal reproductive function in the female rat. In turn, coordinated afferent neuronal signals to GnRH neurons assure the proper timing and amplitude of the preovulatory surge of GnRH. Neurons that originate in the rostral portion of the preoptic area (rPOA) constitute a major afferent input to GnRH neurons by integrating information regarding steroidal milieu and time of day and relaying these signals to GnRH neurons (for review see Ref. 1). It should be noted that considerable confusion exists with regard to the anatomical terminology used to describe the various cell groups found in this region. In the present study, we have adopted the terminology as established by Swanson (2) due to its consistency and detail and define the rPOA as an area that contains all rostral preoptic structures, including most notably the anteroventral periventricular nucleus (AVPV) and rostral portion of the medial preoptic nucleus (rMPN). Evidence that the rPOA in the rodent is a critical site that integrates information on the steroidal milieu and time-of-day comes from several lines of evidence. Electrochemical lesions of the rPOA disrupt the LH surge and the estrous cycle (3). Discrete administration of estradiol specifically within the rPOA induces LH surges (4). Conversely, placement of antiestrogens in this region blocks spontaneous and steroid-induced LH surges (5) and attenuates the rhythm in GnRH messenger RNA (mRNA) and decreases GnRH protein levels (6). It is not clear which specific subdivision of the rPOA mediates the above effects because it is impossible to confine lesions or application of hormones to only the AVPV or rMPN.

A major ongoing interest of neuroendocrine research has been to decipher the roles of various neuropeptides and neurotransmitters in the regulation of GnRH secretion. Neurotensin (NT) is one of the neuropeptides heavily expressed in the rMPN and to a lesser extent in the AVPV, and numerous studies suggest that it mediates the stimulatory effects of estrogen on GnRH secretion. Pharmacological studies have shown that NT amplifies (7, 8) and antisera to NT (9) reduces the magnitude of the LH surge in the rat. In addition, a large proportion of NT-containing neurons in these subdivisions expresses estrogen receptors (10). Consistent with its proposed role in mediating the stimulatory influences of estradiol on GnRH secretion, estradiol stimulates NT gene expression in the rMPN and AVPV (11, 12).

Although NT neurons in the rPOA seem to stimulate GnRH neuronal activity (7, 8), it is not known whether they communicate directly with GnRH neurons. NT-immunoreactive fibers have been observed closely apposed to GnRH neurons in the mouse (13). NT exerts its effects through at least three receptors that have been cloned and designated NT1 (high affinity), nt2, and nt3 (14). Only NT1 is thought to mediate the physiological effects of NT (for review see Ref. 15). Intriguingly, NT1 mRNA is expressed at low to moderate levels around the organum vasculosum of the lamina terminalis (OVLT)/rPOA (16), a region containing a subpopulation of GnRH neurons thought to play an important role in the generation of the LH surge (17).

In the present study, we investigated whether increases in NT gene expression in the rMPN, as assessed by in situ hybridization, may contribute to the occurrence of the preovulatory LH surge on the afternoon of proestrus. We focused on the rMPN because preliminary results demonstrated that only a few neurons in the AVPV express NT mRNA. We reasoned that if NT gene expression influences the cyclic release of LH, we should observe a unique pattern of expression on proestrus, when the LH surge occurs, compared with days when LH secretion is basal. Secondly, we used dual-label in situ hybridization to examine whether, and to what extent, GnRH neurons in the OVLT/rPOA region express NT1 mRNA. In addition, we examined animals at different stages of the estrous cycle to determine whether the extent of colocalization of NT1and GnRH gene expression depends on the stage of the cycle.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult female Sprague Dawley rats (3–4 months of age; Zivic-Miller Laboratories, Inc., Allison Park, PA) were housed under a controlled photoperiod (14-h light, 10-h dark, lights on 0400 h) and provided food and water ad libitum. Vaginal cytology was monitored daily, and only those animals exhibiting at least two consecutive 4-day estrous cycles were used. All procedures were approved by the University of Kentucky Institutional Animal Care and Use Committee.

Groups of rats (n = 6–7) were decapitated at the following time points to monitor NT mRNA levels: 2400 h (diestrous day 2) and 0300, 0800, 1200, 1600, and 2000 h on the day of proestrus; 2400 h on estrus and 1600 h on diestrous day 1. To determine whether NT1 colocalizes with GnRH neurons, rats (n = 4 per group) were killed at 0300 and 1600 h on proestrus, estrus, and diestrous day 1. These time points were chosen to include stages during the estrous cycle in which the steroidal milieu was distinctly different. Furthermore, the preovulatory LH surge occurs exclusively on proestrus.

Tissue preparation
After decapitation, brains were rapidly removed, frozen, and stored at -70 C. Trunk blood was collected, and the serum was frozen until LH and estradiol were measured by RIA. Coronal sections of the brain (12 µm thick) were collected from the region of the hypothalamus containing the organum vasculosum of the lamina terminalis (OVLT) through the rPOA (A 7470–A 6860) (18). Sections were thaw-mounted onto slides and stored at -70 C until they were processed for in situ hybridization.

Probe preparation
The NT riboprobe was generated using a template plasmid containing a 336-bp EcoRV/BglII fragment (nucleotides 626–961) corresponding to the NT-coding domain and proximal 3' untranslated portion of the rat NT/N cDNA (generously provided by Dr. Paul Dobner, University of Massachusetts Medical Center, Worcester, MA). A riboprobe was transcribed in the presence of 50 µM {alpha}-thio-UTP, of which 10%, 25%, or 50% was 35S-labeled. The NT1 riboprobe was generated from a complementary DNA (cDNA) template that was constructed by ligating a 883-bp NcoI/PmlI fragment of the full-length rat NT1 cDNA (kindly provided by Dr. S. Nakanishi, Kyoto University Faculty of Medicine, Kyoto, Japan) into pBluescript II KS+ (Stratagene, La Jolla, CA). This insert corresponds to nucleotides 2481–3364 of the 3' untranslated region of the full-length cDNA. The riboprobe was transcribed in the presence of 100% 35S-{alpha}-thio-CTP. The digoxigenin-labeled GnRH riboprobe was generated by using a 330-bp BamHI/HindIII cDNA fragment corresponding to exons I-IV of GnRH cDNA (generously provided by Dr. P. Seeburg, Max-Planck-Institute for Medical Research, Heidelberg, Germany).

Single-label in situ hybridization
35S-labeled NT. Brain sections from each animal were processed simultaneously in a single-label in situ hybridization assay according to the method of Wise et al. (19) with several modifications. Briefly, sections were fixed in phosphate-buffered 4% paraformaldehyde, treated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0), and dehydrated. Hybridization buffer (50 µl) containing 600 ng/ml labeled NT/N complementary RNA was applied to each slide. Slides were coverslipped and incubated in humid chambers at 45 C for 18 h. Sections were treated with RNase A (25 µg/ml), washed under conditions of increasing stringency, including a 1-h wash at 60 C in 0.2x SSC, dehydrated in ethanol containing 300 mM ammonium acetate, and air-dried. They were dipped in NTB2 emulsion (diluted 1:1 in distilled water), exposed for 15 days at 4 C, developed, and counterstained with cresyl violet.

Dual-label in situ hybridization
35S-labeled NT1 and digoxigenin-labeled GnRH. Sections from each animal were processed simultaneously in a dual-label in situ hybridization following the method of Sannella and Petersen (20) with several modifications. Tissue sections were processed as described above, except that they were also delipidated in chloroform for 5 min. Hybridization buffer (200 µl) containing 4 x 106 cpm of the 35S-CTP-labeled NT1 riboprobe and 2 µl digoxigenin-UTP-labeled GnRH riboprobe was applied to each slide and incubated overnight at 55 C in a humidified chamber. After stringent washes, including a 1-h wash at 63 C in 0.1x SSC, slides were processed for immunocytochemical detection of the digoxigenin-labeled GnRH riboprobe. Briefly, nonspecific binding was prevented by blocking the tissue for 2.5 h in 2x SSC with 0.05% Triton X-100 and 2% normal lamb serum at room temperature. After blocking, slides were washed twice for 10 min each in Buffer A [100 mM Tris HCl (pH 7.5) and 150 mM NaCl] and incubated overnight at 4 C in antidigoxigenin-peroxidase (Roche Molecular Biochemicals, Indianapolis, IN) diluted 1:200 in Buffer A containing 0.3% Triton X-100 and 1% NLS. Sections were rinsed and incubated for approximately 1 h in the chromogen, 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, MO). The DAB solution was prepared by mixing 10 mg DAB in 50 ml 0.1 M Tris (pH 7.6) and adding 8 µl hydrogen peroxide. The reaction was terminated by rinsing in 0.1 M Tris (pH 7.6), followed by distilled water and 70% ethanol. After slides were processed for immunocytochemical detection of the digoxigenin-labeled probe, autoradiographic detection of 35S-labeled probe was carried out as described above. Slides were exposed for 3 weeks at 4 C, developed, and counterstained with Toluidine blue.

Quantitative analysis
The level of NT gene expression in individual cells was quantified using a Bioquant Image Analysis System (R&M Biometrics, Nashville, TN). Two sections containing the rMPN were bilaterally analyzed per animal. All cells within the demarcated area (Fig. 2Go) were included in the analysis. Slides were examined, and a single threshold for determining grains vs. background was set. This threshold setting remained constant throughout the analysis of all slides in this study. In addition, lighting and contrast levels were standardized before taking any measurements to assure that all sections were assessed under the same criteria. The perimeter of each cell was outlined so that the area of the cell covered by silver grains could be measured. Background was determined by taking measurements of unlabeled cells outside the region of interest. A cell was considered labeled if its nucleus was visible within a cluster of silver grains and if the number of silver grains was at least 5x background. Approximately 50 cells per brain section met these criteria.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 2. Schematic drawing depicting the region within the rMPN that was examined for possible changes in NT gene expression over the day of proestrus. OC, Optic chiasm.

 
For the dual-label in situ hybridization, slides were examined under a 40x objective for the presence of silver grains overlying the digoxigenin-labeled GnRH neurons. A GnRH neuron was considered double labeled if GnRH immunoreactivity was present in the same optical plane as the NT1 hybridization signal and if the number of silver grains was at least 3x background.

RIAs
LH. Serum samples were assayed in duplicate using methods similar to those described previously (21). LH-RP-3 was used as the reference material, and iodinated rat LH (Covance Laboratories, Inc., Vienna, VA) as the competitor. The CSU 120 antibody (generously provided by Dr. Terry Nett, Colorado State University, Fort Collins, CO) was used at a dilution of 1:10,000.

17ß-estradiol. Sera were extracted in anhydrous ethyl ether and radioimmunoassayed for 17ß-estradiol concentrations using a double-antibody commercial kit (ICN, Costa Mesa, CA) according to the manufacturer’s directions.

Statistical analysis
Differences among groups were considered significant when P < 0.05. To determine whether a diurnal rhythm in NT gene expression exists on the day of proestrus, one-way ANOVA was performed; post hoc analysis using Duncan’s multiple range test was used to assess which times of day were different from each other. Student’s t test was used to assess whether NT gene expression changed from the morning (2400 h) to afternoon (1600 h) of diestrous day 1. Differences in the number of GnRH mRNA-containing neurons that coexpress NT1 mRNA on different days of the estrous cycle were evaluated by two-way ANOVA, followed by post hoc analysis using Duncan’s multiple range test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distribution of NT mRNA-expressing cells
The distribution of NT mRNA in the AVPV and rMPN subdivisions of the rPOA is shown in Fig. 1Go. The greatest number of NT mRNA-expressing cells was found in the rMPN, with significantly fewer detectable NT mRNA-expressing cells in the AVPV. Increasing the concentration of 35S-UTP (i.e. 10–50%) in the NT ribroprobe did not increase the number of NT mRNA-expressing cells in the AVPV (data not shown). This differential distribution of NT mRNA in the rMPN and AVPV was similar with respect to location and number as that previously reported by Alexander et al. (22).



View larger version (75K):
[in this window]
[in a new window]
  		Figure 1. Darkfield photomicrographs and line drawings of the distribution of NT mRNA-containing cells in the AVPV (A) and the rMPN (B) of the rostral preoptic area in the proestrous rat. NT mRNA is more highly expressed in the rMPN than in the AVPV. Scale bar, 300 µm. OC, Optic chiasm.

 
Levels of NT mRNA in the rMPN on the day of proestrus
We chose to analyze the pattern of NT gene expression in the medial portion of the rMPN (Fig. 2Go) because (1) other laboratories demonstrated that this subpopulation of NT mRNA-containing neurons is sexually dimorphic (22) and varies in intensity over the course of the estrous cycle (22) and that (2) the strongest estrogen-receptive input to the vicinity of the GnRH perikarya in the OVLT/rPOA arises from neurons located in the AVPV as well as the rMPN of the preoptic area (23), suggesting that this population of NT-containing neurons is likely to exert significant effects on the preovulatory surge of GnRH. The level of NT mRNA per cell in the rMPN varied significantly over the day of proestrus (Fig. 3AGo, P < 0.03). NT mRNA levels rose significantly between 2400 h diestrous day 2 and 0300 h proestrus and remained high during the entire day and did not increase further at 1600 h. The darkfield photomicrographs in Fig. 3BGo show labeled cells from representative brain sections from animals killed at 2400 h of diestrous day 2 and at 1600 h of proestrus. They illustrate the increase in the levels of NT mRNA during the day of proestrus. In contrast, no significant difference in NT mRNA per cell was observed on diestrous day 1 when LH concentrations were basal (Table 1Go).



View larger version (90K):
[in this window]
[in a new window]
  		Figure 3. NT mRNA levels in the rMPN in the proestrous rat. A, The level of NT mRNA per cell in the rMPN varied significantly over the day of proestrus. Bars represent mean ± SEM (n = 6–7 rats per group per time point). One-way ANOVA, followed by Duncan’s multiple range test, revealed that NT mRNA levels at 2400 h diestrous day 2 (a) were significantly lower than at all other times on proestrus (P < 0.05). B, Darkfield photomicrographs of representative brain sections from animals killed at 2400 h of diestrous day 2 and at 1600 h of proestrus. Scale bar, 150 µm. 3v, Third ventricle.

 

View this table:
[in this window]
[in a new window]
  		Table 1. NT mRNA/cell (area of enhanced grains/µm2) in the rMPN at 2400 h and 1600 h on proestrus and diestrous day 1

 
Distribution of NT1mRNA-expressing GnRH neurons
We detected many intensely labeled NT1 cells in the medial septum and diagonal band of Broca. In addition, light to moderately labeled cells were localized to the OVLT/rPOA region where the majority of GnRH neurons are found (Fig. 4AGo). The distribution of neurons expressing NT1 mRNA in the rostral forebrain was similar to that reported previously (16).



View larger version (58K):
[in this window]
[in a new window]
  		Figure 4. A, Darkfield photomicrograph showing the distribution of NT1 mRNA-expressing cells in the OVLT/rPOA region. OC, Optic chiasm. Scale bar, 250 µm. B and C, Brightfield photomicrographs of representative sections of the OVLT/rPOA demonstrating GnRH neurons (dark gray precipitate) that express or do not express mRNA for NT1 (silver grains). The arrows depict GnRH mRNA-expressing cells that express mRNA for NT1. Note the GnRH neuron (arrowhead) in B that does not express NT1 mRNA. Scale bar, 15 µm.

 
Dual-label in situ hybridization revealed that NT1 mRNA is expressed in some GnRH neurons in the OVLT/rPOA region (Fig. 4Go, B and C). The percentage of dual-labeled neurons varied significantly depending on the stage of the cycle. Two-way ANOVA revealed a significant interaction between time and day [F (2, 18) = 5.40; P < 0.02]. Further analysis using Duncan’s multiple range test demonstrated that the percentage of GnRH neurons that colocalized with NT1 mRNA was highest on the morning of proestrus compared with all other days of the cycle (Fig. 5BGo). In contrast, the mean number of neurons expressing GnRH mRNA did not differ over the time course studied (Fig. 5AGo).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 5. A, Number of single-labeled GnRH cells and double-labeled cells during different time points of the rat estrous cycle. B, Percentage of GnRH neurons expressing NT1 mRNA in the OVLT/rPOA region during different time points of the rat estrous cycle. Values represent mean ± SEM (n = 4 rats per time point). Two-way ANOVA revealed a significant interaction between time and day [F(2 18 ) = 5.40; P < 0.02]. Further analysis using Duncan’s multiple range test demonstrated that the percentage of GnRH neurons that colocalized with NT1 mRNA was highest on the morning of proestrus (a) compared with all other days of the cycle. Pro, Proestrus; E, estrus; Di, diestrous day 1.

 
Hormone profile
The profiles of serum LH and estradiol concentrations in animals killed on the day of proestrus are presented in Fig. 6Go. LH values were significantly higher at 1600 and 2000 h than at other times of day, and estradiol concentrations showed the expected preovulatory rise on the afternoon of proestrus and returned to basal levels by 2000 h.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 6. Serum estradiol (A) and LH (B) concentrations in proestrous rats. Estradiol levels reach a peak between 1200–1600 h. The preovulatory LH surge reaches a peak at 1600 h.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The findings of the present study demonstrate that the biosynthetic activity of NT-containing neurons in the rMPN increases on proestrous morning, before the preovulatory LH surge. In contrast, we observed no such increase in NT gene expression on diestrous day 1, when the GnRH/LH surge is absent. The correlation of a unique rhythm with the occurrence of the preovulatory LH surge suggests that the increase in NT mRNA is relevant to the generation of the LH surge. Furthermore, we report for the first time that a significant proportion of GnRH neurons in the OVLT/rPOA region express NT1 mRNA and that the number of GnRH neurons that coexpress NT1 mRNA reaches the highest levels on proestrous morning. Taken together, these results suggest that NT neurons in the rMPN play an important role in the neural events responsible for the generation of the preovulatory GnRH surge.

The rPOA contains all rostral preoptic structures including, most notably, the two subdivisions: the AVPV and rMPN. It is well established that the AVPV is a nodal point in the forebrain circuitry essential for regulating reproductive cyclicity in the rat (for review see Ref. 24). On the other hand, the role the rMPN plays in the regulation of cyclic GnRH secretion is not as clear. Simonian et al. (23) recently reported, using retrograde tracing methods, that the strongest estrogen-receptive afferent projection to the vicinity of GnRH cell bodies arises from neurons originating not only in the AVPV but also in the medial portion of the rMPN. Thus, the present results raise the possibility that neurons residing in the rMPN synthesize NT and release the peptide to directly modulate GnRH secretion. Because our data do not provide evidence for a direct anatomical link between NT neurons in the rMPN and GnRH cell bodies in the OVLT/rPOA region, additional studies using anterograde tracing methods will be necessary to thoroughly address this issue.

Our results clearly demonstrate that increased NT gene expression in the rMPN occurs in association with the occurrence of the LH surge. In interpreting these data, we make the assumption that changes in mRNA levels lead to parallel changes in translation of the mRNA and the amount of neuropeptide released. This association between changes of NT mRNA and the release of neuropeptide has not been examined within a single study. Watanobe and Takebe (25) reported that release of NT into the median eminence from cell bodies originating primarily in the arcuate nucleus significantly increases concurrently with the generation of the LH surge. Thus, together, the above study and our current results suggest that NT mRNA expression and neuropeptide secretion are functionally coupled. The exact temporal relationship between NT gene expression and NT peptide release cannot be determined on the basis of these findings. Clarification of this issue will ultimately require analysis of both NT gene expression and release in the same study.

It is interesting that Ciofi (26) recently reported that approximately 30% of all GnRH neurons in the female rat contain NT immunoreactivity. This coexistence of GnRH and NT was observed, for the most part, in neurons encompassing the OVLT/rPOA region. Furthermore, this multipeptidergic neuronal phenotype was not detectable in ovariectomized adult female rats, but became apparent after treatment with a high level of estrogen for 2–7 days. Therefore, it is possible that the NT responsible for regulating cyclical GnRH neuronal activity actually originates in the GnRH cell population itself rather than in the rMPN. However, it should be noted that an autocrine/paracrine role for NT in the regulation of GnRH secretion does not exclude a direct NT input from the rMPN.

The increase in NT gene expression in the rMPN that we observed on proestrus parallels the increase in serum levels of estradiol. Although we did not measure estradiol levels earlier than 2400 h on the morning of proestrus, we (27, 28) and numerous other investigators (29, 30, 31, 32) have shown that serum estradiol gradually increases over diestrous day 2. Thus, it is possible that the changes in NT and NT1 gene expression observed at 0300 h on the morning of proestrus are due to the gradual rise in estradiol on diestrous day 2. Interestingly, Kalra (33) reported that bilateral ovariectomy at 2300 h of diestrous day 2 but not at 0300 h or later on the morning of proestrus prevented the preovulatory surge of LH. Thus, it seems that events mediated by estradiol between 2300 and 0300 h on the morning of proestrus are crucial for the generation of the LH surge.

Several lines of evidence suggest that estradiol may directly stimulate this increase in NT mRNA. First, Alexander et al. (11) demonstrated that administration of estradiol to ovariectomized rats, which results in plasma levels of estradiol within the physiological range, enhances NT mRNA levels in the rPOA within 48 h. In addition, supraphysiological levels of estradiol lead to a striking increase in the number of NT-immunoreactive cell bodies in this same region (12). Second, a substantial population of NT-containing cells in the rPOA expresses estrogen receptors (10). Therefore, estradiol may induce an increase in NT gene expression. Whether the actions of estradiol use the classical mechanism of transactivation, involving nuclear receptor dimerization and binding to consensus estrogen response elements, is unclear since Watters and Dorsa (34) demonstrated that estradiol may induce NT gene expression by influencing the phosphorylation of the cyclic AMP-binding protein.

Our finding that NT1 mRNA colocalizes with GnRH mRNA in the OVLT/rPOA region strongly suggests that NT regulates GnRH secretion by communicating its stimulatory influence directly to GnRH neurons. These findings are consistent with the observation of Hoffman (13) that NT-containing nerve terminals closely appose GnRH cell bodies in the mouse, although the synaptic nature of these appositions has yet to be verified with electron microscopy. The subpopulation of GnRH neurons in the OVLT/rPOA has been considered particularly important in the generation of the LH surge because they (1) exhibit a unique preovulatory and steroid-induced diurnal pattern in GnRH gene expression that is not detectable in GnRH neurons in other regions of the basal forebrain and (2) express c-fos during the preovulatory and steroid-induced LH surge (35, 36, 37, 38). This subpopulation of GnRH neurons may respond to a selected group of neurotransmitters and neuropeptides, including NT, resulting in a cyclical pattern of neuronal activity different from other GnRH neurons. Our observation that the number of GnRH neurons that coexpress NT1 reaches a peak on proestrus is particularly intriguing. This cyclic increase in NT1 in GnRH neurons allows amplification of the impact of the proestrous increase in NT activity on this unique subpopulation of GnRH neurons.

The precise role that NT plays in the regulation of cyclic GnRH secretion on proestrus, as well as the extent to which it interacts with other key neuropeptides and neurotransmitters important for this crucial physiological event, remain to be determined. Because neither infusion (7, 8) nor immunoneutralization (9) of NT influences the timing of the LH surge, one could postulate that the predominant role of NT neurons is to determine the magnitude of the LH surge. Future research is undoubtedly necessary to understand the precise anatomical and functional links that exist between NT neurons and other components of the neural network regulating GnRH secretion.

In summary, levels of NT mRNA in the rMPN significantly increase on the day of proestrus in a manner that suggests that: 1) they are temporally related to changes in the levels of estradiol; and 2) increased NT biosynthetic activity plays an important role in the proestrous GnRH surge. Furthermore, we have demonstrated that a significant number of GnRH neurons express the high-affinity NT receptor and that this expression varies over the estrous cycle. These results, in accordance with previous findings, suggest that NT-containing neurons in the rMPN may be involved in the generation of the LH surge as a result of increased activity and elevated responsiveness of GnRH neurons to stimulation by NT.


   Acknowledgments
 
We thank Kris Krajnak, Dena Dubal, and Katherine Rosewell for superb technical assistance and advice.


   Footnotes
 
1 Supported by NIH Grants AG-02224 and AG-13425 (to P.M.W.) and AG05847 (to M.J.S.). Back

Received November 14, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Herbison AE 1998 Multimodal actions of estrogen. Endocr Rev 19:302–330[Abstract/Free Full Text]
  2. Swanson LW 1987 The hypothalamus. In: Bjorklund A, Hokfelt T (eds) Handbook of Chemical Neuroanatomy. Elsevier, Amsterdam, New York, Oxford, vol 5:1
  3. Wiegand SJ, Terasawa E 1982 Discrete lesions reveal functional heterogeneity of suprachiasmatic structures in regulation of gonadotropin secretion in the female rat. Neuroendocrinology 34:395–404[CrossRef][Medline]
  4. Goodman RL 1978 A quantitative analysis of the physiological role of estradiol and progesterone in the control of tonic and surge secretion of luteinizing hormone in the rat. Endocrinology 102:142–150[Medline]
  5. Petersen SL, Barraclough CA 1989 Suppression of spontaneous LH surges in estrogen-treated ovariectomized rats by microimplants of antiestrogens into the preoptic brain. Brain Res 484:279–289[CrossRef][Medline]
  6. Petersen SL, Cheuk C, Hartman RD, Barraclough CA 1989 Medial preoptic microimplants of the antiestrogen, Keoxifene, affect LHRH mRNA levels, median eminence LHRH concentrations and LH release in ovariectomized, estrogen-treated rats. J Neuroendocrinol 1:279–283[CrossRef]
  7. Ferris CF, Pan JX, Singer EA, Boyd ND, Carraway RE, Leeman SE 1984 Stimulation of luteinizing hormone release after stereotaxic microinjection of neurotensin into the medial preoptic area of rats. Neuroendocrinology 38:144–151
  8. Akema T, Kimura F 1989 Inhibition by catecholaminergic blockers of the neurotensin-induced enhancement of luteinizing hormone secretion in the ovariectomized estrogen-primed rat. Neuroendocrinology 50:688–692[Medline]
  9. Alexander MJ, Mahoney PD, Ferris CF, Carraway RE, Leeman SE 1989 Evidence that neurotensin participates in the central regulation of the preovulatory surge of luteinizing hormone in the rat. Endocrinology 124:783–788[Abstract]
  10. Herbison AE, Theodosis DT 1992 Localization of oestrogen receptors in preoptic neurons containing neurotensin but not tyrosine hydroxylase, cholecystokinin or luteinizing hormone-releasing hormone in the male and female rat. Neuroscience 50:283–298[CrossRef][Medline]
  11. Alexander MJ, Dobner PR, Miller MA, Bullock BP, Dorsa DM, Leeman SE 1989 Estrogen induces neurotensin/neuromedin N messenger ribonucleic acid in a preoptic nucleus essential for the preovulatory surge of luteinizing hormone in the rat. Endocrinology 125:2111–2117[Abstract]
  12. Alexander MJ, Leeman SE 1994 Estrogen-inducible neurotensin immunoreactivity in the preoptic area of the female rat. J Comp Neurol 345:496–509[CrossRef][Medline]
  13. Hoffman GE 1985 Organization of LHRH cells: differential apposition of neurotensin, substance P and catecholamine axons. Peptides 6:439–461[CrossRef][Medline]
  14. Vincent J-P, Mazella J, Kitabgi P 1999 Neurotensin and neurotensin receptors. Trends Pharmacol Sci 20:302–309[CrossRef][Medline]
  15. Rostene WH, Alexander MJ 1997 Neurotensin and neuroendocrine regulation. Front Neuroendocrinol 18:115–173[CrossRef][Medline]
  16. Alexander MJ, Leeman SE 1998 Widespread expression in adult rat forebrain of mRNA encoding high affinity neurotensin receptor. J Comp Neurol 402:475–500[CrossRef][Medline]
  17. Petersen SL, McCrone S, Keller M, Shores S 1995 Effects of estrogen and progesterone on luteinizing hormone-releasing hormone messenger ribonucleic acid levels: consideration of temporal and neuroanatomical variables. Endocrinology 136:3604–3610[Abstract]
  18. Konig JFR, Klippel RA 1963 The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Robert E. Krieger Publishing, Huntington, NY
  19. Wise PM, Scarbrough K, Lloyd JM, Weiland NG, Larson GH, Chiu S 1992 Assessment of proopiomelanocortin gene expression in brain. Methods Neurosci 9:197–208
  20. Sannella MI, Petersen SL 1997 Dual label in situ hybridization studies provide evidence that LHRH neurons do not synthesize mRNA for µ, {kappa}, or {Delta} opiate receptors. Endocrinology 138:1667–1672[Abstract/Free Full Text]
  21. Legan SJ, Callahan WH 1999 Suppression of tonic luteinizing hormone secretion and norepinephrine release near the GnRH neurons by estradiol in ovariectomized rats. Neuroendocrinology 70:237–245[CrossRef][Medline]
  22. Alexander MJ, Kiraly ZJ, Leeman SE 1991 Sexually dimorphic distribution of neurotensin/neuromedin N mRNA in the rat preoptic area. J Comp Neurol 311:84–96[CrossRef][Medline]
  23. Simonian SX, Spratt DP, Herbison AE 1999 Identification and characterization of estrogen receptor {alpha}-containing neurons projecting to the vicinity of gonadotropin-releasing hormone perikarya in the rostral preoptic area of the rat. J Comp Neurol 411:346–358[CrossRef][Medline]
  24. Simerly RB 1998 Organization and regulation of sexually dimorphic neuroendocrine pathways. Behav Brain Res 92:195–203[CrossRef][Medline]
  25. Watanobe H, Takebe K 1993 In vivo release of neurotensin from the median eminence of ovariectomized estrogen-primed rats as estimated by pust-pull perfusion: correlation with luteinizing hormone and prolactin surges. Neuroendocrinology 57:760–764[Medline]
  26. Ciofi P 2000 Phenotypical segregation among female rat hypothalamic gonadotropin-releasing hormone neurons as revealed by the sexually dimorphic coexpression of cholecystokinin and neurotensin. Neuroscience 99:133–147[CrossRef][Medline]
  27. DePaolo LV, Shander D, Wise PM, Barraclough CA, Channing CP 1979 Identification of inhibin-like activity in ovarian venous plasma of rats during the estrous cycle. Endocrinology 105:647–654[Medline]
  28. Wise PM 1986 Effects of hyperprolactinemia on estrous cyclicity, serum luteinizing hormone, prolactin, estradiol, and progesterone concentrations, and catecholamine activity in microdissected brain areas. Endocrinology 118:1237–1245[Abstract]
  29. Kalra SP, Kalra PS 1974 Effects of circulating estradiol during rat estrous cycle on LH release following electrochemical stimulation of preoptic brain or administration of synthetic LRF. Endocrinology 94:845–851[Medline]
  30. Kalra SP, Kalra PS 1974 Temporal interrelationships among circulating levels of estradiol, progesterone and LH during the rat estrous cycle: effects of exogenous progesterone. Endocrinology 95:1711–1718[Medline]
  31. Lu JKH 1983 Changes in ovarian function and gonadotropin and prolactin secretion in aging female rats. In: Meites J (ed) Neuroendocrinology of Aging. Plenum Press, New York, pp 103–122
  32. Smith MS, Freeman ME, Neill JD 1975 The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: prolactin, gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy. Endocrinology 96:219–226[Abstract]
  33. Kalra SP 1975 Observations on facilitation of the preovulatory rise of LH by estrogen. Endocrinology 96:23–28[Abstract]
  34. Watters JJ, Dorsa DM 1998 Transcriptional effects of estrogen on neuronal neurotensin gene expression involve cAMP/protein kinase A-dependent signaling mechanisms. J Neurosci 18:6672–6680[Abstract/Free Full Text]
  35. Lloyd JM, Hoffman GE, Wise PM 1994 Decline in immediate early gene expression in gonadotropin-releasing hormone neurons during proestrus in regularly cycling, middle-aged rats. Endocrinology 134:1800–1805[Abstract]
  36. Rubin BS, Mitchell S, Lee CE, King JC 1995 Reconstructions of populations of LHRH neurons in young and middle-aged rats reveal progressive increases in subgroups expressing fos protein on proestrus and age-related deficits. Endocrinology 136:3823–3830[Abstract]
  37. Hoffman GE, Lee W-S, Attardi B, Yann V, Fitzsimmons MD 1990 Luteinizing hormone-releasing hormone neurons express c-fos antigen after steroid activation. Endocrinology 126:1736–1741[Abstract]
  38. Lee W-S, Smith MS, Hoffman GE 1990 Luteinizing hormone-releasing hormone neurons express Fos protein during the proestrous surge of luteinizing hormone. Proc Natl Acad Sci USA 87:5163–5167[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
S. L. Petersen, E. N. Ottem, and C. D. Carpenter
Direct and Indirect Regulation of Gonadotropin-Releasing Hormone Neurons by Estradiol
Biol Reprod, December 1, 2003; 69(6): 1771 - 1778.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Smith, M. J.
Right arrow Articles by Wise, P. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smith, M. J.
Right arrow Articles by Wise, P. M.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals