Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print May 28, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.017848
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
69/4/1318    most recent
biolreprod.103.017848v1
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Scanlan, N.
Right arrow Articles by Skinner, D. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Scanlan, N.
Right arrow Articles by Skinner, D. C.
Agricola
Right arrow Articles by Scanlan, N.
Right arrow Articles by Skinner, D. C.
BIOLOGY OF REPRODUCTION 69, 1318–1324 (2003)
DOI: 10.1095/biolreprod.103.017848
© 2003 by the Society for the Study of Reproduction, Inc.

Neuroendocrinology

Somatostatin-14 Neurons in the Ovine Hypothalamus: Colocalization with Estrogen Receptor {alpha} and Somatostatin-28(1–12) Immunoreactivity, and Activation in Response to Estradiol

Niamh Scanlan2, Laurence Dufourny3, and Donal C. Skinner1,3

Department of Clinical Veterinary Science,2 University of Bristol, Langford, BS40 5DU, United Kingdom Department of Zoology & Physiology,3 University of Wyoming, Laramie, Wyoming 82071


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pituitary gland growth hormone (GH) secretion is influenced by two hypothalamic neuropeptides: growth hormone-releasing hormone (GHRH) and somatostatin. Recent data also suggest that estrogen modulates GH release, particularly at the time of the preovulatory luteinizing hormone surge, when a coincident surge of GH is observed in sheep. The GHRH neurons do not possess estrogen receptor {alpha} (ER{alpha}), suggesting that estrogen does not act directly on GHRH neurons. Similarly, few somatotropes express ER{alpha}, suggesting a weak pituitary effect of estradiol on GH. It was hypothesized, therefore, that estradiol may affect somatostatin neurons to modulate GH release from the pituitary. Using immunocytochemical approaches, the present study revealed that although somatostatin neurons were located in several hypothalamic sites, only those in the arcuate nucleus (13% ± 2%) and ventromedial nucleus (VMN; 29% ± 1%) expressed ER{alpha}. In addition, we found that all neurons immunoreactive for somatostatin-14 were also immunoreactive for somatostatin-28(1–12). To determine whether increased GH secretion in response to estradiol is through modulation of GHRH and/or somatostatin neuronal activity, a final study investigated whether c-fos expression increased in somatostatin- and GHRH-immunoreactive cells at the time of the estradiol-induced LH surge in intact anestrous ewes. Estradiol significantly (P < 0.05) increased the percentage of GHRH (estradiol, 75% ± 3%; no estradiol, 19% ± 2%) neurons expressing c-fos in the hypothalamus. The percentage of somatostatin-immunoreactive neurons coexpressing c-fos in the estradiol-treated animals was significantly (P < 0.05) higher (periventricular, 44% ± 3%; arcuate, 72% ± 5%; VMN, 81% ± 5%) than in the control animals (periventricular, 22% ± 1%; arcuate, 29% ± 3%; VMN, 31% ± 3%). The present study suggests that estradiol modulates the activity of GHRH and somatostatin neurons but that this effect is most likely mediated through an indirect interneuronal pathway.

estradiol receptor, growth hormone, growth hormone-releasing hormone, mechanisms of hormone action, somatostatin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Evidence is accumulating that growth hormone (GH) plays a critical role in reproduction [1, 2]. Today, GH is commonly used in the treatment of infertility in humans [3, 4], and data suggest that GH acts as a cogonadotropin to enhance the effects of gonadotropins [5]. In hypophysectomized ewes, follicular growth and ovulation were not induced by exogenous gonadotropins unless GH was coadministered [6]. Other reports suggest that GH directly affects gonadotropin responsiveness to GnRH [7–9]. Studies also indicate that estrogen modulates GH secretion. In humans, GH release is elevated around the time of the preovulatory LH surge [10], and a robust GH surge accompanies the estradiol-induced or spontaneous LH surge in ewes [11–13]. Secretion of GH is also increased in postmenopausal women supplemented with estrogen [14, 15], and estradiol administration to ovariectomized ewes is associated with an increase in GH pulse amplitude [16]. The precise mechanism(s) by which estradiol modulates the GH neuroendocrine axis remains poorly understood.

Estradiol may affect two of the principal hypothalamic regulators of GH release: growth hormone-releasing hormone (GHRH) and somatostatin. The term somatostatin refers to a family of peptides: somatostatin-14, somatostatin-28, and somatostatin 28(1–12). These somatostatins are all cleaved from a common, 116-amino acid primary translation product, preprosomatostatin [17, 18]. Somatostatin-28 consists of the entire sequence of somatostatin-14 at its carboxyl terminal, followed by a double pair of basic amino acids and then somatostatin-28(1–12) [19]. Studies in the rat suggest that selective release of somatostatins may occur and that their potency in inhibiting GH secretion from the pituitary is not uniform [19–21]. It is unclear whether somatostatin-28(1–12) and somatostatin-14 operate within the same hypothalamic cells or have a dual function in exerting somatostatin actions in sheep. Some evidence suggests that somatostatin-28(1–12) is preferentially located in neuronal processes in other species [22, 23]. Thus, our first objectives were to determine the distribution of somatostatin-28(1–12) and whether somatostatin-14 and somatostatin-28(1–12) are coexpressed within the same hypothalamic nuclei of the ewe.

We recently reported that although ovine GHRH neurons are surrounded by many {alpha} estrogen receptors (ER{alpha}), GHRH neurons did not express ER{alpha} [11]. If estradiol does not act directly on GHRH neurons, the next obvious hypothesis is that estradiol affects somatostatin secretion to regulate GH release in the ewe. In this context, estradiol would reduce somatostatin secretion into hypophyseal portal blood to cause increased GH release from the pituitary. However, studies in the rat [24] and preliminary investigations in the ewe [25] have found that somatostatin neurons in the periventricular region, which are the major (if not the only) source of somatostatin in the hypophyseal portal system [26], do not express ER{alpha}. Because somatostatin neurons have been shown to synapse on GHRH perikaryia [27, 28], an alternative hypothesis is that estradiol inhibits somatostatin action on GHRH neurons and, thereby, enables increased GHRH and subsequent GH release. In support of this conjecture, a preliminary study suggested that about a third of somatostatin neurons in the ovine ventromedial nucleus (VMN) express ER{alpha} [25]. Thus, our next study sought to confirm the preliminary study in the ewe and to establish whether somatostatin neurons of the periventricular area and VMN express ER{alpha}.

As hypothesized for the effects of estradiol on the GnRH system of the ewe, it is possible that estradiol acts through an interneuronal system to modulate GHRH and somatostatin output. Several studies in ewes have shown that GnRH neurons do not express ER{alpha} [29, 30] but that GnRH perikaryia show increased c-fos expression in response to estradiol [31, 32], where c-fos is used as a marker of neuronal activation. Our final objective, therefore, was to determine whether GHRH and/or somatostatin neurons exhibited increased c-fos expression in response to a GH surge-inducing elevation of estradiol.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

For experiments 1 and 2, sexually mature Dorset horn ewes (n = 5) were ovariectomized under anesthesia, and a Silastic 17ß-estradiol implant (length, 1 cm; outside diameter, 4.64 mm; inside diameter, 3.35 mm) was immediately inserted subcutaneously in the inner aspect of the left forelimb. Ewes were maintained under natural photoperiod; fed daily with hay, straw, and concentrate; and had free access to water. Studies were conducted in November during the breeding season. At the time of the experiment, two progesterone-releasing implants (CIDR; Pharmacia and Upjohn Co., Kalamazoo, MI) were inserted intravaginally for 10 days. The progesterone implants were then removed, and the animals were killed 12 h later. This time corresponds to the early follicular phase in ovary-intact ewes. Brain tissue collected from these ewes was utilized in both experiments 1 and 2.

In experiment 3, sexually mature, ovary-intact Dorset Horn ewes (n = 10) were maintained in outdoor barns under normal photoperiod. Studies were conducted in June, when ewes were anestrous. Previous studies have shown that anestrous ewes show robust GH and LH surges in response to estradiol [12] and exhibit full-amplitude LH surges in response to 50 µg of estradiol injected i.m. [33]. Accordingly, five ewes were injected i.m. with 50 µg of estradiol benzoate (Sigma, St. Louis, MO) in 5 ml of corn oil; the remaining five were injected i.m. with 5 ml of corn oil. Blood samples were collected every 4.5 h, starting from the time of the injections, and all ewes were killed 18 h after the injections. This was the time that we predicted would be the middle of the GH surges in the estradiol-treated animals.

All animals were injected with 25 000 IU of heparin and immediately killed with an overdose of sodium pentobarbitone. All procedures were carried out in accordance with Home Office License No PPL 30/1670.

Tissue Preparation

Animals were decapitated, and the brains were perfused through both carotid arteries with 1 L of 1% sodium nitrite in 0.9% NaCl, followed by 3 L of cold 4% paraformaldehyde and 15% saturated picric acid in 0.1 M phosphate buffer (PB; pH 7.4) and followed by 1 L of 40% sucrose in PB. Following fixation, the brains were dissected out and left in 40% sucrose in PB at 4°C until infiltrated. Six identical sets of coronal sections (thickness, 60 µm; each section within a set was 360 µm apart from the preceding section) were cut on a freezing microtome from the septum through to the caudal hypothalamus at the level of the mammillary bodies. Sections were stored in cryoprotectant [34] at -20°C until immunocytochemistry was performed.

Experiment 1: Localization of Somatostatin-14 and Somatostatin-28(1–12) Immunoreactivity in Specific Hypothalamic Neurons

In the first part of the present study, we sought to determine the distribution of neurons that were immunoreactive for somatostatin 28(1–12) in the ovine hypothalamus. Free-floating sections were removed from cryoprotectant and washed in 0.05 M Tris-buffered saline (TBS; pH 7.8) for 5 min. All washes were repeated three times. Antigens were unmasked according to methods described in detail previously [35]. Briefly, sections were boiled (15 min) in high-temperature antigen-retrieval solution (pH 6.8, 100°C; Vector Laboratories, Burlingame, CA) and left to cool in the unmasking solution (20 min). Sections were washed, immersed in 40% methanol/1% H2O2/TBS solution (10 min), washed, and transferred to 20% normal goat serum/0.1% Triton/TBS for 1 h. Sections were incubated for 72 h in 0.3% Triton X-100 and 5% normal goat serum in TBS containing a polyclonal rabbit anti-somatostatin-28(1–12) antibody (1:2000; AB1752; Chemicon, Temecula, CA). This antibody recognizes somatostatin-28(1–12) as well as somatostatin-28. It should also be noted that at least within the perikaryia, all somatostatin antibodies will also detect preprosomatostatin. At room temperature, sections were washed, incubated in secondary antiserum (1:300, 90 min; biotinylated goat anti-rabbit immunoglobulin [Ig] G; Vector), washed, and placed in streptavidin-horseradish peroxidase (1:100, 90 min; Amersham Pharmacia Biotech, Little Chalfont, U.K.). Visualization of somatostatin-28(1–12) immunoreactivity was performed with diaminobenzidene tetrahydrochloride (DAB) as described previously [35]. Sections were mounted on gelatinized slides, cover-slipped, and analyzed according to the ovine stereotaxic atlas of Richard [36].

To determine whether neurons immunoreactive for somatostatin-14 and somatostatin-28(1–12) were distinctly localized in the ovine hypothalamus, sections were incubated for 72 h in 0.3% Triton X-100 and 5% normal goat serum in TBS containing a monoclonal rat anti-somatostatin antibody (1:250; V1169; Biomeda, Hayward, CA), which recognizes both somatostatin-14 and somatostatin-28, and a polyclonal rabbit anti-somatostatin-28(1–12) antibody (1:500; AB1752). After washing, sections were placed for 90 min in secondary antiserum, fluorescein isothiocyanate-conjugated affinity-pure fab fragment goat anti-rabbit IgG (1:50; Jackson Immunoresearch, West Grove, PA) and Texas Red affinity-pure goat anti-rat IgG secondary antibody (1:50; Jackson), washed, and left in final wash overnight at 4°C. Sections were mounted onto gelatin-coated slides, coverslipped with an anti-fading mounting medium (Vectashield; Vector), and observed using a Leica TCS SP2 confocal microscope.

Experiment 2: Expression of Estrogen Receptors in Somatostatin Neurons

Sections were washed in TBS, unmasked, washed in TBS, and incubated for 72 h at 4°C in a rat monoclonal antibody raised against the human ER{alpha} (2 µg/ml; H222; a gift from Dr. A.E. Herbison, University of Otago, Dunedin, New Zealand). At room temperature, sections were washed, incubated in secondary antiserum (1:300, 90 min; biotinylated goat anti-rat IgG; Vector), washed, and placed in Vectastain Elite kit (1:50, 90 min; Vector). Nuclear immunoreactivity was visualized using nickel-intensified DAB [35].

Sections were washed in TBS, transferred to 40% methanol/0.3% H2O2 TBS for 10 min to deactivate remaining peroxidases, and then placed in one of two rabbit polyclonal anti-somatostatin antibodies, T4103 (1:100; Peninsula Laboratories, San Carlos, CA) or 20067 (1:100; DiaSorin, Wokingham, U.K.), for 72 h. These antibodies recognize both somatostatin-14 and somatostatin-28. At room temperature, sections were washed, incubated in secondary antiserum (1:300, 90 min; biotinylated goat anti-rabbit IgG; Vector), washed, and placed in streptavidin-horseradish peroxidase (1:100, 90 min). Visualization of somatostatin immunoreactivity was performed with DAB as described previously [35]. Sections were mounted on gelatinized slides, coverslipped, and analyzed. Only profiles of neurons in which the nucleus was evident were included in the analysis.

Experiment 3: Activation of Somatostatin and GHRH Neurons by Estradiol

Sections were unmasked, washed in TBS, and incubated for 72 h in 0.3% Triton X-100 and 5% normal goat serum in TBS containing a polyclonal rabbit anti-c-fos antibody (1:1000; sc253; Santa Cruz Biotechnology, Santa Cruz, CA). At room temperature, sections were washed, incubated in secondary antiserum (1:300, 90 min; biotinylated goat anti-rabbit IgG; Vector), washed, and placed in Vectastain Elite kit (1:50, 90 min). Nuclear immunoreactivity was visualized using nickel-intensified DAB.

Sections were washed in TBS, transferred to 40% methanol/0.3% H2O2/TBS for 10 min, and then placed in either rabbit anti-somatostatin (1:100; T4103; Peninsula) or rabbit anti-human GHRH (1:20 000; Peninsula) for 72 h. At room temperature, sections were washed, incubated in secondary antiserum (1:300, 90 min; biotinylated goat anti-rabbit IgG; Vector), washed, and placed in streptavidin-horseradish peroxidase (1:100, 90 min). Visualization of somatostatin or GHRH immunoreactivity was performed with DAB, and sections were mounted on gelatinized slides and coverslipped.

Data were analyzed for each hypothalamic area using the non-parametric Mann-Whitney U-test. Significance was accepted at P < 0.05.

Antibody Specificity

The specificity of the H222 antibody has been described previously [37]. To determine the specificity of the somatostatin-14 antibodies, antibodies were preadsorbed overnight with somatostatin-14, which abolished all specific immunoreactivity. The specificity the somatostatin-28(1–12) antibody was determined by preadsorbing this antibody overnight with somatostatin-28 or somatostatin-14. Somatostatin-28(1–12) immunoreactivity was abolished by somatostatin-28 preadsorption but persisted with somatostatin-14. The c-fos polyclonal antibody was raised against a peptide corresponding to amino acids 128–152, mapping within a highly conserved domain of c-fos of human origin. This sequence is identical to the corresponding mouse, rat, and chicken sequences. The specificity of the GHRH antibody has been previously described [11]. Other controls included omission of the primary antibodies from the incubation procedure and utilization of the inappropriate secondary antisera to visualize the primary antiserum (e.g., goat anti-rabbit with monoclonal rat antiserum). In all cases, the omission of primary antibodies or use of inappropriate secondary antisera resulted in a complete absence of specific immunostaining.

Radioimmunoassay

Plasma samples from experiment 3 were analyzed for LH in duplicate 100-µl aliquots using a previously described radioimmunoassay [38]. The LH concentration was expressed in terms of National Hormone and Pituitary Program ovine-LH 1–4 (courtesy of A.F. Parlow, NIDDK). All LH samples were analyzed in a single assay, and the intraassay coefficient of variation was 5.3%. The assay sensitivity was defined as 2 SD from the zero standard, which was calculated as 0.43 ng/ml.

Plasma GH concentrations were estimated in 100-µl aliquots by a double-antibody RIA and were expressed in terms of National Hormone and Pituitary Program ovine GH-I-5 as previously described [39]. Assay sensitivity was 1.2 ng/ml, and the intraassay coefficient of variation was 11%.

Plasma GH and LH data were analyzed statistically by two-way ANOVA (Graphpad Software, Inc., San Diego, CA). Significance was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1: Localization of Somatostatin-14 and Somatostatin-28(1–12) Immunoreactivity in Specific Hypothalamic Neurons

Somatostatin-28(1–12)-immunoreactive neurons were widely distributed throughout the ovine hypothalamus (Fig. 1), and their distribution corresponded to that previously described for somatostatin-14/28 [40, 41]. In the most rostral regions, somatostatin-28(1–12)-immunoreactive neurons were visible in the caudate nucleus, bed nucleus of the stria terminalis, and the ventrolateral septum (Fig. 1, A and B). Immunoreactive perikaryia were also revealed throughout the medial and lateral preoptic area (Fig. 1A), the nucleus (Fig. 1B), the anterior hypothalamic area (Fig. 1, F and G), and the dorsomedial hypothalamic nucleus and caudal regions of the arcuate nucleus (Fig. 1, E–G). Many somatostatin-immunoreactive neurons were also visible in the VMN (Fig. 1, C–E).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 1. Brain drawings of coronal sections rostral to caudal (A–H) through the hypothalamus of a representative ewe showing the location of somatostatin-28(1–12)-immunoreactive cells. Each dot represents a neuron. 3V, Third ventricle; ac, anterior commissure; ARC, arcuate nucleus; BNST, bed nucleus of the stria terminalis; CN, caudate nucleus; DMN, dorsomedial nucleus; MPOA, medial preoptic area; fx, fornix; mt, mammillary tract

Double-fluorescent immunocytochemistry revealed that all somatostatin-28(1–12)- and somatostatin-14-immunoreactive neurons were expressed within the same hypothalamic nuclei (Fig. 2, A–F). The intensity of labeling did differ between neurons, with some being more intensely immunoreactive for somatostatin-14 and others for somatostatin-28(1–12). Intense immunoreactivity for both somatostatin-14 and somatostatin-28(1–12) was also detected in the external zone of the median eminence (Fig. 2, G and H).



View larger version (109K):
[in this window]
[in a new window]
  		FIG. 2. Somatostatin-14-immunoreactive neurons colocalized with (A–F) somatostatin-28(1–12). All somatostatin-14-immunoreactive neurons in the (A and B) medial preoptic area, (C and D) VMN, and (E and F) arcuate nucleus were also immunoreactive for somatostatin-28(1–12). Also shown are (G) somatostatin-14 and (H) somatostatin-28(1–12) immunoreactive fibers in the external zone of the ovine median eminence. White arrows note somatostatin-immunoreactive neurons. Bar = 56 µm (A and B), 50 µm (C and D), 10 µm (E and F), and 750 µm (G and H)

Experiment 2: Expression of Estrogen Receptors in Somatostatin Neurons

The ER{alpha}-immunoreactive cells were stained black in the nucleus and widely dispersed in the ovine hypothalamus, as previously described [30, 37]. Briefly, ER{alpha}-immunoreactive cells were revealed throughout the central and medial regions of the preoptic area and adjacent to the third ventricle and the organum vasculosum of the lamina terminalis. More ER{alpha}-immunoreactive cells were observed adjoining the anterior hypothalamic area, distinctly in the dorsal anterior hypothalamic area. Many ER{alpha}-immunoreactive cells were also located in the arcuate nucleus and lateral region of the VMN.

Because of the distinct cellular localization of the two antigens, double-labeled neurons were easily observed. No ER{alpha}-immunoreactivity was evident in somatostatin-immunoreactive neurons located in the preoptic area, caudate nucleus, or bed nucleus of the stria terminalis. Only a few ER{alpha}-immunoreactive cells were found surrounding somatostatin neurons of the periventricular nucleus, and no somatostatin-immunoreactive cells in this area of the hypothalamus were found to express ER{alpha} immunoreactivity (Fig. 3A). Some somatostatin-immunoreactive neurons of the VMN expressed nuclear ER{alpha} immunoreactivity (29% ± 2%) (Fig. 3, C and E), and fewer somatostatin-immunoreactive perikaryia in the arcuate nucleus were immunoreactive for ER{alpha} (13% ± 2%) (Fig. 3B). It is of note that with the DiaSorin antibody, we detected few or no somatostatin-immunoreactive perikaryia in the VMN, whereas this population was always visualized with the Peninsula and Biomeda antibodies.



View larger version (102K):
[in this window]
[in a new window]
  		FIG. 3. Immunoreactive somatostatin neurons (brown cytoplasm) colocalized with ER{alpha} (black nuclei). No somatostatin neurons in the (A) periventricular area were immunoreactive for ER{alpha}. A single immunoreactive ER{alpha} cell is noted by the asterisk. In the (B) arcuate nucleus and (C and D) VMN, some somatostatin neurons were immunoreactive for ER{alpha}. Also shown is (E) a photomicrograph showing two somatostatin-immunoreactive neurons in the VMN that did not express ER{alpha}. Black arrows note ER{alpha} colocalized with somatostatin, whereas white arrows show somatostatin neurons that do not express ER{alpha}. Bar = 40 µm (A), 18 µm (B), 15 µm (C), and 14 µm (D and E)

Experiment 3: Activation of Somatostatin and GHRH Neurons by Estradiol

One ewe that was administered estradiol did not show an increase in either LH or GH secretion and was excluded from the study. Previous studies from our laboratory [42] have shown that some animals take longer to respond to estradiol with an LH surge. Estradiol administration to the remaining four anestrous ewes caused a significant (P < 0.005) increase in GH and LH secretion (Fig. 4A) in comparison to the five ewes that received no estradiol.



View larger version (89K):
[in this window]
[in a new window]
  		FIG. 4. A) GH and LH concentrations in anestrous ewes that were injected with either estradiol or vehicle. Estradiol (E) caused a significant (P < 0.005, two-way ANOVA) rise in both GH and LH levels in comparison to vehicle-treated ewes. The c-fos-immunoreactivity (black nuclei) was evident in both (B and C) somatostatin and (D) GHRH neurons (brown cytoplasm). Not all (C) somatostatin and (E) GHRH neurons expressed c-fos. Black arrows note c-fos colocalized with somatostatin or GHRH, whereas white arrows show somatostatin or GHRH neurons that do not express c-fos. Bar = 10 µm (B and E), 12 µm (C), and 23 µm (D)

Intensely black-stained, c-fos-immunoreactive nuclei were observed throughout the ovine hypothalamus. Somatostatin-immunoreactive neurons expressed c-fos in both estradiol-treated and control animals (Fig. 4, B and C). However, the percentage of somatostatin-immunoreactive neurons coexpressing c-fos in the estradiol-treated animals was significantly (P < 0.05) higher (periventricular, 44% ± 3%; arcuate, 72% ± 5%; VMN, 81% ± 5%) than in the control animals (periventricular, 22% ± 1%; arcuate, 29% ± 3%; VMN, 31% ± 3%).

Similarly, GHRH-immunoreactive neurons expressed c-fos nuclear staining (Fig. 4D) in both the estradiol-treated and control animals. However, the percentage of GHRH-immunoreactive neurons coexpressing c-fos in the estradiol-treated animals was significantly (P < 0.05) higher (75% ± 3%) than in the control animals (19% ± 2%).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our study confirms an earlier, preliminary investigation that VMN, but not periventricular, somatostatin neurons colocalize with ER{alpha} [25]. In addition, the present study describes, to our knowledge for the first time, the distribution of somatostatin-28(1–12)-immunoreactive cells in the ovine hypothalamus. More importantly, it reveals that all neurons immunoreactive for somatostatin-14 in the ewe are also immunoreactive for somatostatin-28(1–12), suggesting that these two somatostatin peptides are not selectively produced by neurons within the ovine hypothalamus. Finally, the present study provides compelling evidence that two of the neurotransmitter systems known to control GH secretion are activated in response to an estradiol treatment, which induces both GH and LH surges.

The wide distribution of somatostatin-immunoreactive neurons throughout the ovine hypothalamus that we found in the present study, with specific concentrations in the preoptic area, periventricular region, arcuate nucleus, and VMN, is similar to the distribution reported for somatostatin-14, which would include somatostatin-28, in earlier studies in the ewe [40, 41]. This distribution also appears to be comparable to that described in several other mammalian species, including, among others, dog [43], rat [40, 44], hedgehog [41], and pig [45]. In contrast to some earlier studies in the monkey and rat [22, 23], we found no evidence in the ewe for selective immunoreactivity for somatostatin-28(1–12) in the neuronal processes as opposed to the cell body. It is noteworthy that the DiaSorin antibody was unable to detect the VMN population of somatostatin neurons, which is clearly evident in other studies of the ewe [25] and is also evident following in situ hybridization [46]. This DiaSorin antibody was also used in studies of the bovine hypothalamus, and notably, no somatostatin-immunoreactive neurons were detected in the VMN of that species [45].

Extremely few studies have investigated whether all the somatostatins derived from preprosomatostatin are differentially produced or secreted by the hypothalamus. Reports in rats indicate that somatostatin-28 may be selectively more potent than somatostatin-14 at inhibiting GH release from the anterior pituitary [20, 21], whereas others suggest that there may be selective release of somatostatin-14 and somatostatin-28 from the hypothalamus [19]. We found that both somatostatin-14 and somatostatin-28(1–12) were detectable within the external zone of the median eminence region, suggesting that these somatostatins are not selectively secreted. The initial objective of the present study was to determine whether selective localization of somatostatin-14 and somatostatin-28(1–12) immunoreactivity occurred in the ovine hypothalamus. If so, we planned to conduct double-labeling studies to establish whether ER{alpha} expression was greater in one of the subpopulations. Because we found that all somatostatin-14 neurons were also immunoreactive for somatostatin-28(1–12) in the ovine hypothalamus, these studies were obviously unnecessary.

As indicated by previous studies in the ewe, the main sites of ER{alpha}-immunoreactive cells were the preoptic area, anterior hypothalamic area, VMN, and arcuate nucleus [30, 37]. Our study supports an earlier, preliminary investigation in the ewe, which reported that more than a third of somatostatin-immunoreactive neurons of the VMN express ER{alpha} [25]. Similarly, in the rat, a high proportion of VMN somatostatin neurons were reported to express ER{alpha} [24]. Several studies report that estrogen influences somatostatin in the VMN of the rat [47] and guinea pig [48]. In ovariectomized guinea pigs, estradiol treatment increased the number of somatostatin-immunoreactive neurons in all regions of the VMN and caused a small increase in the arcuate nucleus [48]. Other studies, using in situ hybridization, reported that estrogen increases somatostatin synthesis in both the periventricular region and VMN of the rat hypothalamus [47, 49, 50].

In contrast to the VMN, somatostatin neurons of the periventricular nucleus, which are thought to be the major source of somatostatin in the hypophyseal portal system [26, 51], do not express ER{alpha}. This finding is not surprising, because few immunoreactive ER{alpha} cells have been reported in the periventricular region of the ewe [30, 37]. Our finding is consistent with an earlier, preliminary study in sheep [25] and has also been reported in the rat [24]. Similarly, periventricular somatostatin neurons in the guinea pig were devoid of progesterone receptors [52]. Thus, if only the periventricular somatostatin population projects to the hypophyseal portal vasculature of the sheep, then the effects of estradiol on somatostatin secretion likely are indirect and mediated through an interneuronal system. Bluet-Pajot et al. [26] hypothesized that somatostatin neurons in the arcuate nucleus/VMN region could project to the periventricular area and, thereby, influence somatostatin secretion in the hypophyseal portal system. To our knowledge, this elegant hypothesis remains to be tested in the ewe, but it could be a pathway through which estradiol may modulate hypophyseal portal somatostatin levels.

It is also possible that estradiol modulates GH release through a different neuronal system. In this context, the recently discovered hypothalamic peptide ghrelin, which displays potent GH-releasing activity (for review, see [53]), could be targeted by estradiol. Indeed, recent studies in rodents [54] suggest that ghrelin neurons are located in close proximity to GHRH and somatostatin neurons. Ghrelin given intravenously specifically releases GH from the pituitary, whereas the GH-releasing activity of ghrelin in vivo is dependent on GHRH [55]. However, it is worth noting that intracerebroventricular administration of ghrelin stimulated GH release but inhibited LH secretion [56]. In contrast, the present study confirmed our previous investigation of ovariectomized ewes during the breeding season [11], and the results of others [12, 13], which showed that estradiol stimulates both GH and LH. Nevertheless, this interesting hypothesis that estradiol may target ghrelin to influence GH secretion at the time of the LH surge is worthy of further investigation.

Whether another neuronal system that can influence GH secretion directly is involved is not known. However, both somatostatin and GHRH neurons clearly are significantly activated by a surge-inducing treatment of estradiol. It is noteworthy that one animal, which did not show an increased secretion of GH or LH in response to estradiol, also did not show elevated c-fos levels in GHRH or somatostatin neurons. Because a few of the somatostatin neurons in the arcuate nucleus and VMN were found to express ER{alpha}, a proportion of the somatostatin neurons in these areas may have been activated directly by estradiol. Nevertheless, all GHRH neurons [11] and the majority of somatostatin neurons were not found to express ER{alpha}, so it is apparent that activation of these neurons must be primarily indirect. Evidence indicates that synaptic communication occurs between GHRH and somatostatin neurons [27]; thus, activation of one GH-regulating system may lead to activation of the other. This ability of estradiol to increase c-fos immunoreactivity in GHRH and somatostatin neurons at the time of the GH/LH surge is markedly similar to the results of studies on the GnRH system of the ewe [31, 32]. These studies reported increased c-fos-related antigen immunoreactivity in GnRH perikaryia, which was abolished when no surge occurred [31]. Further studies are required to establish what happens to GHRH and somatostatin in the hypophyseal portal blood at the time of the estradiol-induced GH surge.

In summary, we have found that preprosomatostatin does not appear to be preferentially cleaved into either somatostatin-14 or somatostatin-28(1–12) in the ovine hypothalamus. Moreover, only somatostatin neurons in the arcuate nucleus and VMN colocalize with ER{alpha}, whereas somatostatin neurons in the periventricular nucleus, which are thought to project to the hypophyseal portal system, do not express ER{alpha}. Despite the general paucity of ER{alpha} expression in somatostatin neurons and their complete absence in GHRH neurons [11], both these GH-regulating neurotransmitter systems showed signs of increased activity at the time of estradiol-induced GH surge. Whether an overlap occurs between the neuronal systems transducing the effects of estradiol to the GnRH neurons and the GHRH/somatostatin neurons remains to be determined.


   ACKNOWLEDGMENTS
 
We thank Dr. Allan Herbison (University of Otago, Dunedin, New Zealand) for his generous gift of the H222 antibody.


   FOOTNOTES
 
1 Correspondence: Donal C. Skinner, Department of Zoology & Physiology, University of Wyoming, Laramie, WY 82071. FAX: 307 766 5625; dcs{at}uwyo.edu Back

Received: 3 April 2003.

First decision: 20 April 2003.

Accepted: 8 May 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bachelot A, Monget P, Imbert-Bollore P, Coshigano K, Kopchick JJ, Kelly PA, Binart N. Growth hormone is required for ovarian follicular growth. Endocrinology 2002 143:4104-4112[Abstract/Free Full Text]
  2. Codner E, Cassorla F. Growth hormone and reproductive function. Mol Cell Endocrinol 2002 186:133-136[CrossRef][Medline]
  3. Homburg R, Eshel A, Abdalla HI, Jacobs HS. Growth hormone facilitates ovulation induction by gonadotrophins. Clin Endocrinol (Oxf) 1988 29:113-117[Medline]
  4. Blumenfeld Z, Amit T. The role of growth hormone in ovulation induction. Ann Med 1994 26:249-254[Medline]
  5. Childs GV. Growth hormone cells as cogonadotropes: partners in the regulation of the reproductive system. Trends Endocrinol Metab 2000 11:168-175[CrossRef][Medline]
  6. Eckery DC, Moeller CL, Nett TM, Sawyer HR. Localization and quantification of binding sites for follicle-stimulating hormone, luteinizing hormone, growth hormone, and insulin-like growth factor I in sheep ovarian follicles. Biol Reprod 1997 57:507-513[Abstract]
  7. Hugues J, Torresani T, Herve F, Martin-Pont B, Tamboise A, Santarelli J. Interest of growth hormone-releasing hormone administration for improvement of ovarian responsiveness to gonadotropins in poor responder women. Fertil Steril 1991 55:945-951[Medline]
  8. Franks S. Growth hormone and ovarian function. Baillieres Clin Endocrinol Metab 1998 12:331-340[Medline]
  9. Zachmann M. Interrelationships between growth hormone and sex hormones—physiology and therapeutic consequences. Horm Res 1992 38:1-8
  10. Faria ACS, Bekenstein LW, Booth RA, Vaccaro VA, Asplin CM, Veldhuis JD, Thorner MO, Evans WS. Pulsatile growth hormone release in normal women during the menstrual cycle. Clin Endocrinol 1992 36:591-596[Medline]
  11. Scanlan N, Skinner DC. Estradiol modulation of growth hormone secretion in the ewe: no growth hormone-releasing hormone neurons and few somatotropes express estradiol receptor {alpha}. Biol Reprod 2002 66:1267-1273[Abstract/Free Full Text]
  12. Landefeld TD, Suttie JM. Changes in messenger ribonucleic acid concentrations and plasma levels of growth hormone during the ovine estrous cycle and in response to exogenous estradiol. Endocrinology 1989 125:1474-1478[Abstract]
  13. Malven PV, Haglof SA, Jiang H. Serum concentrations of luteinizing hormone, growth hormone, and prolactin in untreated and estradiol-treated ovariectomized ewes after immunoneutralization of hypothalamic neuropeptide Y. J Anim Sci 1995 73:2105-2112[Abstract]
  14. Friend KE, Hartman ML, Pezzoli SS, Clasey JL, Thorner MO. Both oral and transdermal estrogen increase growth hormone release in postmenopausal women–a clinical research center study. J Clin Endocrinol Metab 1996 81:2250-2256[Abstract]
  15. Genazzani A, Gamba O, Nappi L, Volpe A, Petraglia F. Modulatory effects of a synthetic steroid (tibolone) and estradiol on spontaneous and GH-RH-induced GH secretion in postmenopausal women. Maturitas 1997 28:27-33[CrossRef][Medline]
  16. Dutour A, Briard N, Guillaume V, Magnan E, Cataldi M, Sauze N, Oliver C. Another view of GH neuroregulation: lessons from the sheep. Eur J Endocrinol 1997 136:553-565[Medline]
  17. Patel YC, O'Neil W. Peptides derived from cleavage of prosomatostatin at carboxyl- and amino-terminal segments. Characterization of tissue and secreted forms in the rat. J Biol Chem 1988 263:745-751[Abstract/Free Full Text]
  18. Rabbani SN, Patel YC. Peptides derived by processing of rat prosomatostatin near the amino-terminus: characterization, tissue distribution, and release. Endocrinology 1990 126:2054-2061[Abstract]
  19. Epelbaum J. Somatostatin in the central nervous system: physiology and pathological modification. Prog Neurobiol 1986 27:63-100[CrossRef][Medline]
  20. Brazeau P, Ling N, Esch F, Bohlen P, Benoit R, Guillemin R. High biological activity of the synthetic replicates of somatostatin-28 and somatostatin-25. Regul Pept 1981 1:255-264[CrossRef][Medline]
  21. Tannenbaum GS, Ling N, Brazeau P. Somatostatin-28 is longer acting and more selective than somatostatin-14 on pituitary and pancreatic hormone release. Endocrinology 1982 111:101-107[Abstract]
  22. Lewis DA, Campbell MJ, Morrison JH. An immunohistochemical characterization of somatostatin-28 and somatostatin-28(1–12) in monkey prefrontal cortex. J Comp Neurol 1986 248:1-18[CrossRef][Medline]
  23. Morrison JH, Benoit R, Magistretti PJ, Bloom FE. Immunohistochemical distribution of pro-somatostatin-related peptides in cerebral cortex. Brain Res 1983 262:344-351[CrossRef][Medline]
  24. Herbison AE, Theodosis DT. Absence of estrogen receptor immunoreactivity in somatostatin (SRIF) neurons of the periventricular nucleus but sexually dimorphic colocalization of estrogen receptor and SRIF immunoreactivities in neurons of the bed nucleus of the stria terminalis. Endocrinology 1993 132:1707-1714[Abstract]
  25. Herbison AE. Neurochemical identity of neurones expressing oestrogen and androgen receptors in the sheep hypothalamus. J Reprod Fertil Suppl 1995 49:371-383
  26. Bluet-Pajot MT, Epelbaum J, Gourdji D, Hammond C, Kordon C. Hypothalamic and hypophyseal regulation of growth hormone secretion. Cell Mol Neurobiol 1998 18:101-123[CrossRef][Medline]
  27. Horvath S, Palkovits M, Gorcs T, Arimura A. Electron microscopic immunocytochemical evidence for the existence of bidirectional synaptic connections between growth hormone-releasing hormone- and somatostatin-containing neurons in the hypothalamus of the rat. Brain Res 1989 481:8-15[CrossRef][Medline]
  28. Liposits Z, Merchenthaler I, Paull WK, Flerko B. Synaptic communication between somatostatinergic axons and growth hormone-releasing factor (GRF) synthesizing neurons in the arcuate nucleus of the rat. Histochemistry 1988 89:247-252[CrossRef][Medline]
  29. Lehman MN, Karsch FJ. Do gonadotropin-releasing hormone, tyrosine hydroxylase-, and ß-endorphin-immunoreactive neurons contain estrogen receptors? A double-label immunocytochemical study in the Suffolk ewe. Endocrinology 1993 133:887-895[Abstract]
  30. Herbison AE, Robinson JE, Skinner DC. Distribution of estrogen receptor-immunoreactive cells in the preoptic area of the ewe: colocalization with glutamic acid decarboxylase but not luteinizing hormone-releasing hormone. Neuroendocrinology 1993 57:751-759[Medline]
  31. Richter TA, Robinson JE, Evans NP. Progesterone treatment that either blocks or augments the estradiol-induced gonadotropin-releasing hormone surge is associated with different patterns of hypothalamic neural activation. Neuroendocrinology 2001 73:378-386[CrossRef][Medline]
  32. Moenter SM, Karsch FJ, Lehman MN. Fos expression during the estradiol-induced gonadotropin-releasing hormone (GnRH) surge of the ewe: Induction in GnRH and other neurons. Endocrinology 1993 133:896-903[Abstract]
  33. Clarke IJ. Gonadotrophin-releasing hormone secretion (GnRH) in anoestrous ewes and the induction of GnRH surges by oestrogen. J Endocrinol 1988 117:355-360[Abstract]
  34. Watson RE, Weigand SJ, Clough RW, Hoffman G. Use of a cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology. Peptides 1986 7:155-159[Medline]
  35. Skinner DC, Caraty A, Allingham R. Unmasking the progesterone receptor in the preoptic area and the hypothalamus of the ewe: no colocalisation with gonadotropin-releasing neurons. Endocrinology 2001 142:573-579[Abstract/Free Full Text]
  36. Richard P. Atlas Stereotaxique du Cerveau de Brebis "Prealpes du Sud.". Paris: Editions INRA; 1967
  37. Lehman MN, Ebling FJP, Moenter SM, Karsch FJ. Distribution of estrogen receptor-immunoreactive cells in the sheep brain. Endocrinology 1993 133:876-886[Abstract]
  38. Niswender GD, Reichert LE, Midgley AR, Nalbandov AV. Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology 1969 84:1166-1173[Medline]
  39. Jaffe CA, Huffman BW, Demott-Friberg R. Insulin hypoglycemia and growth hormone secretion in sheep: a paradox revisited. Am J Physiol 1999 277:E253-E258
  40. Willoughby JO, Oliver JR, Fletcher TP, Clarke IJ. Distribution of somatostatin immunoreactivity in sheep hypothalamus: a comparison with that of the rat. Arch Histol Cytol 1995 58:31-36[Medline]
  41. Papadopoulos GC, Karamanlidis AN, Dinopoulos A, Antonopoulos J. Somatostatin-like immunoreactive neurons in the hedgehog (Erinaceus europaeus) and the sheep (Ovis aries) central nervous system. J Comp Neurol 1986 244:174-192[CrossRef][Medline]
  42. Skinner DC, Harris TG, Evans NP. The duration and amplitude of the luteal phase progesterone increment times the estradiol-induced LH surge in ewes. Biol Reprod 2000 62:1135-1142[Abstract/Free Full Text]
  43. Hoffman GE, Hayes TA. Somatostatin neurons and their projections in dog diencephalon. J Comp Neurol 1979 186:371-391[CrossRef][Medline]
  44. Kawano H, Daikoku S, Saito S. Immunohistochemical studies of intrahypothalamic somatostatin-containing neurons in rat. Brain Res 1982 242:227-232[CrossRef][Medline]
  45. Leshin LS, Barb CR, Kiser TE, Rampacek GB, Kraeling RR. Growth hormone-releasing hormone and somatostatin neurons within the porcine and bovine hypothalamus. Neuroendocrinology 1994 59:251-264[Medline]
  46. Bruneau G, Tillet Y. Localization of the preprosomatostatin mRNA by in situ hybridization in the ewe hypothalamus. Peptides 1998 19:1749-1758[CrossRef][Medline]
  47. Zorrilla R, Simard J, Rheaume E, Labrie F, Pelletier G. Multihormonal control of pre-pro-somatostatin mRNA levels in the periventricular nucleus of the male and female rat hypothalamus. Neuroendocrinology 1990 52:527-536[Medline]
  48. Dufourny L, Warembourg M. Estrogen modulation of neuropeptides: somatostatin, neurotensin and substance P, in the ventrolateral and arcuate nuclei of the female guinea pig. Neurosci Res 1999 33:223-228[CrossRef][Medline]
  49. Baldino F, Jr. Fitzpatrick-McElligott S, O'Kane TM, Gozes I. Hormonal regulation of somatostatin messenger RNA. Synapse 1988 2:317-325[CrossRef][Medline]
  50. Werner H, Koch Y, Baldino F Jr, Gozes I. Steroid regulation of somatostatin mRNA in the rat hypothalamus. J Biol Chem 1988 263:7666-7671[Abstract/Free Full Text]
  51. Bluet-Pajot MT, Durand D, Drouva SV, Mounier F, Pressac M, Kordon C. Further evidence that thyrotropin-releasing hormone participate in the regulation of growth hormone secretion in the rat. Neuroendocrinology 1986 44:70-75[Medline]
  52. Dufourny L, Warembourg M. Colocalization of progesterone receptor and somatostatin immunoreactivities in the hypothalamus of the male and female guinea pig. Neuroendocrinology 1996 64:215-224[Medline]
  53. Petersenn S. Growth hormone secretagogues and ghrelin: an update on physiology and clinical relevance. Horm Res 2002 58:suppl 356-61[Abstract/Free Full Text]
  54. Cowley MA, Smith RG, Diano S, Tschop M, Pronchuk N, Grove KL, Strasburger CJ, Bidlingmaier M, Esterman M, Heiman ML, Garcia-Segura LM, Nillni EA, Mendez P, Low MJ, Sotonyi P, Friedman JM, Liu H, Pinto S, Colmers WF, Cone RD, Horvath TL. The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis. Neuron 2003 37:649-661[CrossRef][Medline]
  55. Kamegai J, Tamura H, Shimizu T, Ishii S, Sugihara H, Oikawa S. Regulation of the ghrelin gene: growth hormone-releasing hormone upregulates ghrelin mRNA in the pituitary. Endocrinology 2001 142:4154-4157[Abstract/Free Full Text]
  56. Furuta M, Funabashi T, Kimura F. Intracerebroventricular administration of ghrelin rapidly suppresses pulsatile luteinizing hormone secretion in ovariectomized rats. Biochem Biophys Res Commun 2001 288:780-785[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Toxicol SciHome page
G. Giusi, R. M. Facciolo, M. Canonaco, E. Alleva, V. Belloni, F. Dessi'-Fulgheri, and D. Santucci
The Endocrine Disruptor Atrazine Accounts for a Dimorphic Somatostatinergic Neuronal Expression Pattern in Mice
Toxicol. Sci., January 1, 2006; 89(1): 257 - 264.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
T. Yonezawa, K. Mogi, J. Y. Li, R. Sako, K. Yamanouchi, and M. Nishihara
Modulation of Growth Hormone Pulsatility by Sex Steroids in Female Goats
Endocrinology, June 1, 2005; 146(6): 2736 - 2743.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
D. Pillon, A. Caraty, C. Fabre-Nys, D. Lomet, M. Cateau, and G. Bruneau
Regulation by Estradiol of Hypothalamic Somatostatin Gene Expression: Possible Involvement of Somatostatin in the Control of Luteinizing Hormone Secretion in the Ewe
Biol Reprod, July 1, 2004; 71(1): 38 - 44.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
J. H. Sliwowska, H. J. Billings, R. L. Goodman, L. M. Coolen, and M. N. Lehman
The Premammillary Hypothalamic Area of the Ewe: Anatomical Characterization of a Melatonin Target Area Mediating Seasonal Reproduction
Biol Reprod, June 1, 2004; 70(6): 1768 - 1775.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
69/4/1318    most recent
biolreprod.103.017848v1
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Scanlan, N.
Right arrow Articles by Skinner, D. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Scanlan, N.
Right arrow Articles by Skinner, D. C.
Agricola
Right arrow Articles by Scanlan, N.
Right arrow Articles by Skinner, D. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS