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Neuroendocrinology |
Unité Mixte de Recherche, Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique/Centre National de la Recherche Scientifique/Université François Rabelais de Tours, 37380 Nouzilly, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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estradiol, gonadotropin-releasing hormone, hypothalamus, luteinizing hormone, somatostatin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Within the mediobasal hypothalamus (MBH) of the ewe, numerous neurons synthesizing somatostatin are present in the infundibular nucleus (IN) and the ventrolateral division of the ventromedial nucleus (VMN) [5, 6]. The role of somatostatin in the regulation of GH secretion has been well-established in rats as in other species [7]. However, the physiological role of the somatostatin-containing neurons located in the VMN remains undefined. In the ewe, the MBH, which contains a dense population of neurons expressing estradiol-receptor alpha (ER
) [8], constitutes the major site of action for estradiol to regulate the induction of both the preovulatory GnRH surge and sexual behavior [9, 10]. Colocalization studies have shown that about 70% of the neurons expressing estrogen receptors in the VMN synthesize somatostatin [11], and 13% of the somatostatinergic neurons located in the IN express ER [12]. Moreover, in ewes injected intramuscularly with estradiol to induce preovulatory GnRH and LH surges, it was observed that the percentage of somatostatin-immunoreactive neurons coexpressing c-fos during the surge was significantly higher in the VMN and the IN in the estradiol-treated animals than in control animals, which received only the vehicle [12]. Therefore, the cells synthesizing somatostatin located in the VMN and/or the IN might play a role in the induction of the preovulatory GnRH surge or sexual behavior.
In ovariectomized ewes treated with progesterone and estradiol, recent studies have demonstrated that an elevated level of estradiol for only a few hours is sufficient to activate the pathways that will lead to the stimulation of both the preovulatory-like GnRH and LH surges and sexual behavior 12–16 h later [13, 14]. However, the phenotypes of these estradiol-sensitive cells of the MBH and the nature of the neurotransmitters or neuromodulators synthesized in the MBH during the short time of estradiol stimulation remain to be clearly defined. Therefore, because of the possible role of the steroid-receptive somatostatin neurons located in the VMN and the IN in inducing preovulatory GnRH surge and/or sexual behavior, the first goal of the present experiment was to analyze the early effect of estradiol on the expression of the preprosomatostatin (PPS) gene. Using in situ hybridization, we assessed the variations of the PPS mRNA in the MBH of ovariectomized Ile de France ewes subjected to a short estradiol treatment (4 h), which was previously determined as sufficient to induce preovulatory surge and estrous behavior [15, 16].
Following this, we wanted to test the effect of somatostatin on LH secretion. Several studies in the rat have indicated that somatostatin does not affect gonadotropin release [1, 17]. However, it was previously suggested that sufficiently large somatostatin doses could block the release of all pituitary hormones [18], and an in vitro study demonstrated that somatostatin is able to decrease the amount of GnRH released in perifusion of mediobasal hypothalamic slices in rat [19]. As no data exist in sheep, we examined whether intracerebroventricular (ICV) administration of somatostatin could influence LH secretion in ewes. We also evaluated if this treatment could modify pituitary GH secretion.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental procedures were performed in accordance with local animal regulations (Authorization No. 37801 of the French Ministry of Agriculture).
The experiment was conducted using ovariectomized Ile de France ewes during the breeding season, according to a model of preovulatory surge induction that simulates the hormonal variations occurring during a natural sexual cycle (Fig. 1A). This model is known to induce repeatedly the preovulatory GnRH and LH surges [20]. Mature ewes showing regular cycles were ovariectomized and immediately treated with a 10-mm subcutaneous Silastic implant containing estradiol (17ß-estradiol; Sigma Chemical Co., L'Isle d'Abeau Chesnes, France) and an intravaginal progesterone-releasing controlled internal drug-release dispenser (Inter AG, Hamilton, New Zealand). After 12 days, the progesterone implants were removed to simulate luteal regression, and 22 h later, 4 x 30-mm subcutaneous estradiol implants were inserted into the thigh of each ewe for 4 h. Animals were then tested to check that all of them responded to this short estradiol signal by developing a preovulatory LH surge and estrous behavior, as in natural hormonal conditions. To achieve this, jugular blood samples were collected from each ewe every 2 h over a 24-h period starting at the time of estradiol implant insertion, and LH was measured. An LH surge was defined when LH concentrations were superior to twice the basal level and to 10 ng/ml for at least two consecutive samples. Estrous behavior was evaluated 18–22 h after estradiol administration by counting the frequency of immobilizations shown by a ewe in response to the courtship of a ram during a test period [21]. A ewe was considered to be showing estrous behavior when the percentage of immobilizations exceeded 50% of the total number of courtship interactions.
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Two days after this preliminary experiment, animals showing estrous behavior as well as an LH surge were again treated for 12 days with a vaginal progesterone implant. Animals were then subjected to an experimental procedure corresponding to either an in situ hybridization experiment (n = 12) or an ICV infusion experiment (n = 9).
In Situ Hybridization Experiment
Experimental procedure Twenty-two hours after progesterone removal, the ewes were divided into two groups: stimulated ewes (n = 6) received 4 x 30-mm estradiol implants, while control ewes (n = 6) received 4 x 30-mm empty implants (Fig. 1B). Four hours later, ewes were killed by decapitation and the brains were perfused with 2 L of physiological saline containing 1% sodium nitrite followed by 4 L of 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. Hypothalami were dissected, postfixed in the same solution at 4°C for a further 24 h, and immersed in a 15% saccharose solution at 4°C. Three days later, the tissue blocks were frozen in dry ice after Tissue-Tek inclusion and stored at –80°C until sectioning. Fifteen micrometer-thick coronal sections through the MBH were cut on a cryostat and stored at –20°C until in situ hybridization analysis was performed. Every 25th section was counterstained with cresyl violet to determine the neuroanatomical location of the labeling.
Radioactive in situ hybridization The antisense and sense probes used were synthesized from the PPS cDNA cloned by Bruneau and Tillet [6]. The pKS-bluescript vector containing the complete ovine coding sequence PPS cDNA was linearized by EcoRI or HindIII and used as a template to synthesize antisense or sense 35S-UTP-labeled cRNA probes, respectively, using the in vitro transcription kit (Stratagene, La Jolla, CA). Probe labeling and radioactive in situ hybridization were performed according to the protocol described by Bruneau and Tillet [6], except that 250 000 cpm/ section were applied in a 40-µl volume and a 25-µg/ml RNase A treatment was performed before thorough washing.
The slides were briefly dehydrated by successive baths in 30%, 50%, 70%, 95%, and 100% ethanol; air dried; and dipped in Kodak NTB2 photographic emulsion (Eastman Kodak, Rochester, NY) half-diluted in sterile water. After 2 wk storage at 4°C, the emulsion-coated slides were developed in Kodak D-19, fixed in 30% (w/v) sodium thiosulfate, counterstained with neutral red, and cover slipped for microscope analysis.
Detection of PPS mRNA-containing cells An in situ hybridization experiment was performed in the most heavily labeled region of the MBH containing the VMN and the IN, and corresponding to the plate C previously described by Bruneau and Tillet [6] in their Figure 4. Five hypothalamic sections of this part of the MBH (Fig. 2A) were hybridized with the antisense probe for each of the 12 ewes, and one section from each ewe was tested with the sense probe. Alternate sections were selected from all the sections stored in order to have close but nonadjacent sections. The hybridized sections were used to determine the mean number of silver grains per cell in the VMN and the IN. As the density of cells was too high to detect individually all the labeled neurons, the mean numbers of somatostatinergic cells per section were not evaluated. The cellular PPS mRNA levels were assessed by analyzing silver grain density over individual cells using a Biocom image analyzer (Explora Nova, La Rochelle, France). With this system, the operator outlined the silver grain cluster over each cell, and a silver grain density was determined. A mean area for detecting the mean number of silver grains per cell was established and this standardized area was transposed on each somatostatinergic cell to detect a constant cytoplasmic area for counting. No fewer than 70 and 30 hybridized cells were counted per ewe for the VMN and the IN, respectively, and the mean numbers of silver grains per neuron were established for each ewe. Counting was done blinded toward the treatment for each section.
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Statistical analysis An average value was obtained for each ewe for PPS gene expression. As independent hybridization experiments were conducted for each pair of ewes (for each experiment: an estradiol-treated ewe and a control ewe), statistical analysis was carried out using a paired Student t-test, with P < 0.05 being considered significant.
Intracerebroventricular Infusion Experiment
At least 2 months in advance of experimentation, each of the nine experimental ewes had a permanent guide cannula stereotaxically introduced into the third ventricle according to the protocol previously described in Skinner et al. [22]. During the experiment, ewes were housed in individual pens, fed maintenance rations of fodder, and given free access to water. Blood sampling was from an indwelling jugular venous canula inserted at least 12 h before the start of the experiment. For the experiment and 22 h after the removal of progesterone, a polyethylene catheter (outer diameter, 0.7 mm; inner diameter, 0.3 mm; Biotrol, Paris, France) was inserted through the guide cannula so that the distal tip ended at the tip of the guide cannula. Animals then received ICV via the catheter connected to a portable syringe pump (Graseby Medical, Watford, Hertfordshire, UK), physiological saline for 5 h (vehicle, 180 µl per h; period P1), and then somatostatin diluted in physiological saline for the following 5 h (180 µl and 90 µg/h; period P2; Sigma). Blood samples were collected at 10-min intervals from 0700 h to 1700 h during the infusion, and also from 1700 h to 2200 h after the end of any infusion (period P3; Fig. 1C).
Hormone assays Blood samples were assayed for LH in duplicate 100-µl aliquots of plasma using a previously described RIA method [23, 24]. All samples from the experiment were run in a single assay. The intraassay coefficient of variation averaged 8%.
Plasma GH concentrations were measured in duplicate using a RIA kit (ovine GH-oGH-I-5 and ovine GH antiserum-anti-oGH-3) provided by the National Hormone and Pituitary Program (National Institutes of Health) according to the supplied protocol. All samples were processed in a single assay with an intraassay coefficient of variation of 9%.
Analysis of LH plasma concentrations The LH data were analyzed with the Munro algorithm [25], which is a modified version of the Pulsar algorithm [26]. The nadir and amplitude for each pulse and the mean nadir and mean pulse amplitude for each ewe were calculated by the program.
Statistical analysis A repeated measures analysis of variance (ANOVA) was used to compare the hormone concentrations profiles between the three periods of time. An error degree of 5% was considered as significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A 4-h treatment of 4 x 30-mm estradiol implants applied in the test cycle induced an LH surge, as well as estrous behavior in all ewes (mean time of the LH surge onset: 15 ± 1 h after estradiol administration; mean amplitude of the LH surge: 32.4 ± 2.4 ng/ml; the receptivity index reached 100% between 18 and 22 h after estradiol administration for all ewes).
Effect of Estrogen Treatment on PPS mRNA Expression
Four hours of estradiol significantly increased the mean number (± SEM) of silver grains per somatostatin expressing-cell by about 50% in the VMN and 42% in the IN (P < 0.01; Figs. 2 and 3). In control and estradiol-treated ewes, respectively, the densities detected were about 84 ± 15 and 126 ± 22 silver grains per somatostatinergic neuron in the VMN, and about 69 ± 9 and 98 ± 13 in the IN.
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Effect of ICV Infusion of Somatostatin on Plasma LH Pulsatility and Concentrations
Intracerebroventricular administration of somatostatin totally inhibited the pulsatility of LH secretion within 83.3 min (± 7.3) and dramatically reduced the mean basal level of LH secretion (Table 1 and Figs. 4 and 5). Comparing LH concentrations and pulsatility in P2 with P1 or P3, we detected a specific effect with the infusion of somatostatin (ANOVA, P < 0.01). Moreover, comparing P1 and P3, we observed that neither the infusion of physiological saline nor the time of the day (morning or evening) modified LH secretion (ANOVA, P > 0.05).
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Effect of ICV Infusion of Somatostatin on GH Secretion
As shown in Figure 6, fluctuations in GH pulsatile secretion were observed over the 15-h period. However, no effect of ICV somatostatin infusion could be detected on GH secretion, with close plasma concentrations for P1, P2, and P3 (mean GH concentrations for P1, P2, and P3, respectively [± SEM]: 2.8 ± 0.4 ng/ml, 3.1 ± 0.9 ng/ml, and 2.9 ± 0.2 ng/ml; ANOVA, P > 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in ewes [11, 12], and about 70% of the neurons expressing ER in this region synthesize somatostatin [11]; there is a strong suggestion that these somatostatinergic neurons are directly regulated by estradiol. In the ovine IN, although no ER-containing neurons synthesizing somatostatin were initially detected [11], a recent study identified a small subpopulation of ER-containing neurons colocalizing somatostatin [12]. Therefore, this result establishes an anatomic support for a direct effect of estradiol in the somatostatinergic neurons of the IN too. However, as this subpopulation seems to be relatively restricted (13% of the somatostatin-containing neurons of the sheep IN express ER) and because no ERß was detected in the sheep IN [27], the variation of PPS mRNA expression observed in our study in the IN could also result from an indirect action of estradiol via interneurons or from another estradiol-induced regulatory pathway not mediated by classic nuclear ER and ERß. Our study was performed on a model of ovariectomized ewes subjected to hormonal treatments known to induce both preovulatory surge and sexual behavior. Our result focuses on a crucial point defined by the 4-h estradiol treatment, which may be the moment for the activation or inhibition of neurotransmitter or neuromodulator gene expression in the MBH for the induction of these two events [15, 16]. To the best of our knowledge, data about somatostatin and central control of the reproductive function are relatively limited. Concerning the preovulatory surge, it has been observed in the ewe that the percentage of somatostatin-immunoreactive neurons coexpressing c-fos in estradiol-treated animals was significantly higher in the VMN and the IN than in control animals during the preovulatory GnRH and LH surges [12]. Together, this observation and our result allow us to hypothesize that somatostatin may play a role in the mechanisms involved in the regulation of the preovulatory surge by estradiol. Moreover, some anatomical results favor the involvement of this neuropeptide in the induction of the preovulatory surge. In the ewe, both retrograde and anterograde tracing studies have shown projections from the lateral part of the VMN to the rostral preoptic area in the vicinity of GnRH neuron perikarya [28, 29]. The identity of these neuronal pathways has not yet been determined, but some fibers containing somatostatin have recently been observed in contact with GnRH neuron perikarya in the preoptic area using confocal microscopy analysis (unpublished results).
We demonstrated that ICV administration of somatostatin, which simulates the increase in PPS mRNA expression observed by in situ hybridization after the estradiol treatment, totally inhibited the pulsatility of LH secretion. The same effect has previously been observed in the ovariectomized female rat [30]. In vitro studies seem to exclude a direct action of somatostatin on the pituitary gland, with a diffusion of somatostatin to the pituitary via the median eminence analogous to the effect of ICV injections [31]. In this way, Yu et al. [32] did not find any effect of somatostatin on basal release of LH. However, somatostatin may act at the hypothalamic level to block neuronal GnRH secretion, as Rotsztejn et al. [19] showed that somatostatin inhibits in vitro release of GnRH from rat mediobasal hypothalamic slices. Moreover, Van Vugt et al. [33] demonstrated that centrally applied somatostatin inhibits the LH surge in female rats, with a percentage of c-fos-containing GnRH cells decreasing after somatostatin treatment compared with control treatment. Therefore, our neurophysiological results support a role of somatostatin in the control of LH secretion via the GnRH neurons. The precise site and mechanisms of this action remain to be defined.
We cannot rule out that the modifications in the expression of PPS mRNA that we observed could be related to the regulation of GH secretion. Estradiol modulates GH release particularly at the time of the preovulatory LH surge, where a coincident surge of GH is observed in sheep [34–37]. We found no effect of ICV somatostatin administration on GH secretion. This result is in agreement with data showing that acute immunization against somatostatin in sheep does not lead to an increase in basal GH secretion or modification to GH nycthemeral rhythms [38]. Moreover, this also indicates that the effects observed on LH secretion are not due to a general activation at the brain centers by somatostatin.
The increase of PPS mRNA expression coincides with the time of the negative feedback that estradiol exerts in the central nervous system during the preovulatory period [39] and closely precedes the induction of sexual behavior. Moreover, as preovulatory estradiol replacement by somatostatin ICV infusion abolished LH pulsatility and dramatically decreased the mean basal level of LH secretion, we hypothesized that somatostatin may be involved in the negative feedback of estradiol.
As the MBH constitutes the major site of action for estradiol to regulate the induction of both the preovulatory GnRH surge and sexual behavior in the ewe [9, 10], the variations of PPS mRNA observed could also be linked to the control of sexual behavior that occurs at the time of the LH preovulatory surge. However, data about the role of somatostatin in sexual behavior induction are lacking. Only one study has suggested the possible involvement of somatostatinergic neurons in the regulation of sexual behavior in the female rat, as they demonstrated that the somatostatinergic neurons in the VMN are activated after mating in females with sexual dimorphism [40].
In summary, in the model of ovariectomized ewes submitted to sequential hormonal treatment by progesterone and estradiol to induce preovulatory surge, we demonstrated that a short estradiol treatment is sufficient to increase the PPS mRNA amount in the MBH. This area of the brain is the primary central site for estradiol action to induce both negative and positive feedback on GnRH and LH secretion. In a similar experimental model with pretreatment by progesterone, somatostatin was intracerebroventricularly infused instead of giving estradiol implants. We determined an inhibitory effect of ICV-infused somatostatin on LH secretion and presumably on GnRH secretion, which can simulate the negative feedback of estradiol occurring at the beginning of the follicular phase before the surge. Therefore, we can hypothesize that the increase in PPS mRNA after a 4-h estradiol treatment could be involved in the negative feedback of estradiol on GnRH and LH secretion.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received: 9 October 2003.
First decision: 31 October 2003.
Accepted: 9 February 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(ER) and ERß messenger ribonucleic acid in the preoptic area and hypothalamus of the sheep: comparison of males and females. Endocrinology 2000 141:2951-2962This article has been cited by other articles:
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  S. Ben Said, D. Lomet, D. Chesneau, L. Lardic, S. Canepa, D. Guillaume, C. Briant, C. Fabre-Nys, and A. Caraty Differential Estradiol Requirement for the Induction of Estrus Behavior and the Luteinizing Hormone Surge in Two Breeds of Sheep Biol Reprod, April 1, 2007; 76(4): 673 - 680. [Abstract] [Full Text] [PDF]
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 J. Robinson Prenatal programming of the female reproductive neuroendocrine system by androgens. Reproduction, October 1, 2006; 132(4): 539 - 547. [Abstract] [Full Text] [PDF]
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C. J. McManus, R. L. Goodman, N. V. Llanza, M. Valent, A. B. Dobbins, J. M. Connors, and S. M. Hileman Inhibition of Luteinizing Hormone Secretion by Localized Administration of Estrogen, but not Dihydrotestosterone, Is Enhanced in the Ventromedial Hypothalamus During Feed Restriction in the Young Wether Biol Reprod, October 1, 2005; 73(4): 781 - 789. [Abstract] [Full Text] [PDF]
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