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Biol Reprod 2004, 10.1095/biolreprod.104.028936
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BIOLOGY OF REPRODUCTION 71, 813–819 (2004)
DOI: 10.1095/biolreprod.104.028936
© 2004 by the Society for the Study of Reproduction, Inc.

Neuroendocrinology

Centrally Applied Somatostatin Inhibits the Estrogen-Induced Luteinizing Hormone Surge via Hypothalamic Gonadotropin-Releasing Hormone Cell Activation in Female Rats1

Harmke H. Van Vugt2,3, Hans J.M. Swarts3, Bert J.M. Van de Heijning3, and Eline M. Van der Beek3,4

Human and Animal Physiology Group,3 Animal Sciences Group, Wageningen University, 6709 PJ, Wageningen, The Netherlands Numico Research,4 Department of Biomedical Research, 6700 CA, Wageningen, The Netherlands


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Overexpression of growth hormone (GH) as well as GH-deficiency dramatically impairs reproductive function. Decreased reproductive function as a result of altered GH release is, at least partially, due to changes at the hypothalamic-pituitary level. We hypothesize that hypothalamic somatostatin (SOM), the inhibiting factor of GH release from the pituitary, may play a central role in the "crosstalk" between the somatotropic and gonadotropic axes. In the present study we investigated the possible effects of a centrally applied SOM analog on the LH surge and the concurrent activation of hypothalamic GnRH neurons in female rats. To this end, female rats were treated with estradiol 2 wk after ovariectomy and were given a single central injection with either the SOM analog, octreotide, or saline just prior to surge onset, after which hourly blood samples were taken to measure LH. Two weeks later, the experimental setup was randomly repeated to collect brains during the anticipated ascending phase of the LH surge. Vibratome sections were subsequently double-stained for GnRH and cFos peptide. Following octreotide treatment, LH surges were significantly attenuated compared to those in saline-treated control females. Also, octreotide treatment significantly decreased the activation of hypothalamic GnRH neurons. These results clearly demonstrate that SOM is able to inhibit LH release, at least in part by decreasing the activation of GnRH neurons. Based on these results, we hypothesize that hypothalamic SOM may be critically involved in the physiological regulation of the proestrus LH surge.

gonadotropin-releasing hormone, growth hormone, hypothalamus, luteinizing hormone, ovulatory cycle


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Growth hormone (GH) secretion from the pituitary is regulated by the inhibitory neurohormone somatostatin (SOM), released from neurons located in the periventricular nucleus of the hypothalamus (PeVN), and by the stimulating effects of GH-releasing hormone (GHRH), released from neurons located in the arcuate nucleus (ARC) of the hypothalamus [1, 2]. It is well known that the functionality of the somatotropic and gonadotropic axes are closely related. Overexpression of GH as well as GH deficiency causes dramatic changes in reproductive function (i.e., a reduced fertility in animals that overexpress GH [3–5] and complete infertility in GH-deficient animals [4, 6]). At the level of the ovaries, GH overexpression results in only primary, secondary, and atretic follicles, and no corpora lutea [7], whereas GH-deficient animals are hypogonadal [6].

Apart from effects on gonadal function, GH also appears to affect reproduction at the hypothalamic and pituitary levels.

Steroid hormone (i.e., estradiol [E2] and progesterone) production in the gonads is regulated by the gonadotropins LH and FSH, which are secreted from the pituitary, and in turn are stimulated by GnRH released from the hypothalamus. Both LH and FSH stimulate several processes in the gonads [8]. In male Ames dwarf mice lacking GH, plasma gonadotropin levels are reduced [6] and the GnRH-stimulated LH synthesis and secretion in vitro is decreased [9]. On the other hand, overexpression of GH in male transgenic mice results in decreased LH and FSH mRNA levels in the pituitary, in a decreased GnRH secretion from the hypothalamus in vitro, and in attenuated GnRH-induced LH and FSH responses in vitro [6, 10]. Also, in male GH-receptor knockout mice, in which GH levels are consistently elevated, the LH response to a GnRH stimulus is attenuated [5, 6]. In female transgenic mice overexpressing GH, the proestrus LH surge is decreased [6] and the distribution of GnRH-containing neurons in the hypothalamus is altered [7]. These data show that decreased reproductive function as a result of altered GH release is, at least partially, characterized by changes at the hypothalamic-pituitary level.

Pre-pro SOM mRNA levels in the PeVN are decreased in GH-deficient animals [11], whereas the number of SOM cells in the PeVN is elevated in animals overexpressing GH [7]. Because SOM, which originates from the PeVN, inhibits GH secretion from the pituitary, and as changes in the somatotropic axis clearly result in changes in the gonadotropic axis, which are at least partially evoked at the hypothalamic-pituitary level, we hypothesize that SOM may play a central role in the "crosstalk" between these two axes.

Indeed, SOM has been shown to affect the reproductive axis at the level of the pituitary: in male rats, SOM inhibits the GnRH-induced release of LH in vitro [12] and decreases plasma LH in vivo [13]. Moreover, multiple central injections with SOM or an SOM analog resulted in smaller and pycnotic gonadotropic cells and in decreased gonadotropic cell numbers in the pituitary of both male and female rats [13–16].

These studies show that chronic high concentrations of SOM can inhibit LH release, probably by affecting the gonadotropic cells in the pituitary. However, based on the data from animals that overexpress or lack GH, we hypothesize that SOM may also affect the reproductive axis within the hypothalamus.

In the present study, we aimed to obtain more insight into the possible direct effects of SOM at the level of the hypothalamus on the regulation of the LH surge in female rats. To this end, we studied the LH release from the pituitary as well as the activation of hypothalamic GnRH cells, following a single central injection with the SOM analog octreotide just prior to surge onset in ovariectomized (OVX) E2-treated rats.


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

Seventeen female Wistar rats (Harlan, Horst, The Netherlands), 10 wk of age on arrival, were individually housed in a room with controlled lighting (12L:12D, lights-on at 0200 h, Zeitgebertime [ZT] 0), temperature (21°C), and humidity (70%), and with standard pelleted food and tap water available ad libitum. A dim red light remained on at all times, including the dark period. The Wageningen University Animal Ethics Committee approved all experiments.

Experimental Design

Prior to surgery, rats were anesthetized by an i.p. injection with a mixture of Ketamine (Kombivet, Etten-Leur, The Netherlands) and Rompun (Bayer AG, Leverkusen, Germany) (respectively, 16.7% and 60% diluted in saline; 1 ml/kg body weight [BW]). All animals underwent bilateral ovariectomy (Day 1) and received an indwelling jugular vein catheter directed toward the right atrium for the stress-free collection of blood samples. In addition, an intracerebroventricular (i.c.v.) guide cannula was placed in the right lateral ventricle of the brain.

On Days 12 and 13 after ovariectomy, all rats received an s.c. injection with estradiol benzoate (EB; 12.5 µg/0.1 ml oil) at 0800 h (ZT 6) to induce an LH surge on Day 14.

On Day 14, rats received an i.c.v. injection with either the SOM analog octreotide (Sandostatine; Novartis Pharma B.V., Arnhem, The Netherlands; 1.0 µg/2 µl; n = 10) or saline (control; 2 µl; n = 7) at 0800 h (ZT 6). Octreotide is a synthetic octapeptide derivative of the natural SOM. The pharmaceutical effects of octreotide are similar to those of SOM, but the effects last longer. Octreotide is used as a treatment for patients with acromegaly and for patients with symptoms associated with functional gastro-entero-pancreatic endocrine tumors, and it clearly inhibits GH secretion [17]. Blood samples were collected every hour from 0830 h (ZT 6.5) to 1730 h (ZT 15.5) for determination of plasma LH concentrations. In addition, from 1030 h (ZT 8.5) to 1130 h (ZT 9.5), samples were drawn every 12 min for GH determination. Samples were centrifuged and the plasma was diluted in 0.02 M phosphate buffer (pH 7.4; 1:4 dilution for LH and 1:5 dilution for GH) and stored at –20°C until assay.

After a recovery period of 3 wk, all rats again received an s.c. injection of EB at 0800 h (ZT 6) on two consecutive days. On the following day, the animals were randomly given an i.c.v. injection with either octreotide (n = 8) or saline (n = 9) at 0800 h (ZT 6), after which all animals were perfused between 1330 and 1400 h (ZT 11.5 and 12), just prior to the onset of the dark period.

Prior to perfusion, animals were given an overdose of Nembutal anesthesia (CEVA sante animale B.V., Maassluis, The Netherlands; 1.5 ml/ kg BW, i.p.), after which a blood sample was taken directly from the heart for plasma LH determination. Subsequently, each rat was perfused transcardially with 200 ml of saline followed by 300 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (4% PFA; pH 7.4). Brains were removed from the skull immediately after perfusion and postfixed in the same fixative for 25 h at 4°C. Prior to sectioning, tissue blocks containing the hypothalamus were embedded in 20% gelatin and fixed with 4% PFA for 4 h at 4°C [18]. Sections of 40 µm were sliced using a Vibratome (Leica, Solms, Germany) and were stored in 0.1 M Tris buffer containing 0.9% NaCl (TBS; pH 7.4) at 4°C until further use.

Immunocytochemistry

One-third of the coronal brain sections of each animal was double-stained for GnRH and cFos peptide using free-floating immunocytochemistry techniques (see also [19]). Sections were pretreated with 3% H2O2 (Merck, Darmstadt, Germany) in TBS for 30 min, followed by extensive washing with TBS.

For the cFos staining, sections were incubated with the primary polyclonal rabbit antibody against cFos (SC-052, #C076; Santa Cruz Biotechnology, Santa Cruz, CA; final dilution 1:10 000) diluted in supermix (TBS containing 0.5% Triton-X-100 and 0.25% gelatin) for 1 h at room temperature, followed by 4 nights at 4°C. This was followed by incubation with biotinylated goat anti-rabbit immunoglobulin G (GaR-bio; Vector Laboratories, Burlingame, CA; 1:400 in supermix) for 1.5 h at room temperature, and Avidin-Biotin Complex-elite (ABC; Vector Laboratories; final dilution 1:1200 in supermix) for 2 h at room temperature. Between incubation steps, sections were thoroughly washed with TBS. Immunoreactivity of cFOS was visualized by incubation with 0.05% 3,3'-diaminobenzidine (DAB; Sigma Chemical Company, St. Louis MO) in TBS containing 0.2% nickelammoniumsulphate and 0.03% H2O2 for 20 min.

For the double staining, cFos-stained sections were washed in a graded series of methanol, including 100% methanol with 0.3% H2O2 (short wash steps, each lasting 5 min; 45 min total). After extensive washing with TBS, the sections were incubated with the primary polyclonal rabbit antibody against GnRH (PLR 005; Eurodiagnostics, Apeldoorn, The Netherlands; final dilution 1:10 000 in supermix) for 1 night at room temperature. This was followed by incubation with GaR-bio (1:400 in supermix) for 1.5 h at room temperature, and ABC (final dilution 1:1200 in supermix) for 2 h at room temperature. Between incubation steps, sections were thoroughly washed with TBS. GnRH immunoreactivity was visualized by incubation with 0.05% DAB in TBS containing 0.1% imidazole and 0.03% H2O2 for 8 min.

Stained sections were mounted on albumin-coated slides, dried, dehydrated, and coverslipped in DEPEX (BDH Laboratory Supplies, Poole, England). Activated (cFos-immunoreactivity-containing) and nonactivated GnRH-immunoreactive (GnRH-ir) neurons were counted in different areas of the hypothalamus using a light microscope.

Radioimmunoassay

LH and GH were determined in a double-antibody radioimmunoassay for rat-LH (see also [20]) or rat-GH using materials supplied by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK; Bethesda, MD) (for LH, rLH-I-9 as label and anti-rLH-s-11 as antiserum; for GH, rGH-I-6 as label and anti-rGH-S-5 as antiserum), and using Sac-cel (donkey anti-rabbit; Welcome Reagents, Beckenham, U.K.) as a second antibody. The levels of LH and GH are expressed in terms of NIDDK-rLH-RP-2 and NIDDK-rGH-RP-2, respectively. The assay sensitivity was 0.2 ng/tube for LH and 0.9 ng/tube for GH at the 90% B/B0 binding level. The intraassay variation was determined using pooled rat serum and was 8.5% for both LH and GH.

Data Analysis

To determine the effects of the SOM analog octreotide on LH release, surge characteristics of each saline- or octreotide-treated rat were compared. These included 1) baseline levels as determined in blood samples collected at 0830 h (ZT 6.5), 2) the time of peak onset defined as the time at which plasma LH levels had increased to twice the baseline level and remained at this level or higher, 3) the time of peak LH levels (i.e., the time at which the highest hormone levels were determined), 4) the magnitude of the peak LH release (ng/ml plasma at peak time), 5) the number of animals that showed an LH peak (incidence of peak), and 6) the total amount of LH released, determined as the cumulative value of hormone levels during the entire sampling period (area under the curve; AUC). To include data from animals that did not show samples with values higher than twice the baseline level (i.e., n = 1 in the saline group and n = 7 in the octreotide group), for statistical analysis the parameters peak onset, peak time, and peak height were chosen to be the value of the last sample. Three animals (n = 1 in the saline group and n = 2 in the octreotide group) showed several samples, although not successive, in which LH was increased to higher than twice the baseline level. From these animals, the sample with the highest value was decided to be the peak onset, as well as peak time and peak height.

To determine the effect of the SOM analog on GH release, the total amount of GH released (i.e., the cumulative value of hormone levels during the complete sampling period [AUC]), was compared between saline- and octreotide-treated animals.

Brain sections were divided into three separate areas, based on the distribution of GnRH cells [19, 21]; that is, the diagonal band of broca (DBB) (area B, two to six sections), the organum vasculosum of the lamina terminalis (OVLT) (area C, three to six sections), and the medial preoptic area (MPO) (area D, three to five sections). These are the areas containing the GnRH neurons that are predominantly responsible for the generation of the GnRH surge, inducing the preovulatory LH surge [22]. In these areas, the total number of GnRH-ir cells as well as the number of activated GnRH-ir cells (i.e., GnRH cells containing a cFos-positive nucleus) was determined.

Data were analyzed using the SPSS statistical analysis system (SPSS, Chicago, IL). To compare basal LH levels, peak LH value, both LH and GH AUC, and perfusion LH values between saline- and octreotide-treated rats, separate one-way analyses of variance were used. To compare LH surge onset time and LH peak time between treatments, Mann-Whitney U-tests were used, and for the incidence of an LH peak, a Fisher exact test was used. Furthermore, to compare both the total number of GnRH-ir cells and the number of activated GnRH-ir cells between the two treatments, one-way analyses of variance were used. Differences were considered significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Octreotide Treatment on LH and GH Release

Repeated EB treatment induced small but significant LH surges in six out of seven OVX animals that were i.c.v. injected with saline. LH levels started to rise at ZT 11.5 ± 0.8 and peaked at ZT 12.3 ± 0.7, with an average LH peak height of 3.24 ± 0.66 ng/ml (Table 1). Variations in peak onset as well as peak time and height were comparable to previous studies using this animal model [20, 23].


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  		TABLE 1. LH surge characteristics in estrogen-treated ovariectomized animals

A single i.c.v. injection with the SOM analog octreotide significantly suppressed the EB-induced LH surge (Fig. 1). Octreotide had no effect on basal plasma LH levels, but significantly decreased LH peak height, AUC, and the incidence of an LH peak. Also, the onset time of the LH peak and the LH peak time were significantly delayed after treatment with the SOM analog (Table 1). Moreover, plasma LH values at the time of perfusion (ZT 11.5–12.0) were significantly lower in octreotide-treated rats than in saline-treated rats (2.7 ± 0.6 and 5.5 ± 0.9, respectively; P = 0.02), demonstrating that the effect of the central octreotide treatment on LH release was similar in both experimental parts.



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  		FIG. 1. Mean ± SEM plasma LH concentrations in OVX+EB rats that received a single i.c.v. injection with saline (open circles; n = 7) or the SOM analog octreotide (closed circles; n = 10)

To test the physiological significance of octreotide, we also measured plasma GH levels. GH secretion patterns showed high individual variations in the saline-treated rats. Not all control rats showed a clear GH peak in this short period of sampling. However, we found an overall lowering of plasma GH levels after octreotide treatment (i.e., the GH AUC was significantly lower in octreotide-treated animals compared to saline-treated animals) (Fig. 2).



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  		FIG. 2. Plasma GH concentrations of all individual OVX+EB rats that received either a single i.c.v. injection with saline (open squares; n = 7) or the SOM analog octreotide (closed squares; n = 10) and the total amount of GH released during the complete sampling period (AUC; insert; light bar: saline-treated, dark bar: octreotide-treated). AUC (mean ± SEM) was significantly decreased in octreotide-treated rats compared to saline-treated rats (P = 0.000)

Effect of Octreotide Treatment on Hypothalamic GnRH Cells

The total number of GnRH-ir cells in the brain areas DBB (area B), OVLT (area C), and MPO (area D) of OVX+EB rats was slightly increased in octreotide-treated rats compared to saline-treated rats (values were 168 ± 12 and 146 ± 12, respectively), albeit not significant. The total percentage of activated (cFos-ir containing) GnRH-ir neurons was significantly lower in octreotide-treated OVX+EB rats than in saline-treated OVX+EB rats (Fig. 3). Although the percentage of activated GnRH-ir neurons after octreotide treatment was reduced in all areas (B–D) when evaluated per area, the difference compared to saline-treated rats was statistically significant only in area C. Percentages in saline- and octreotide-treated rats were, respectively, 2.7 ± 1.6 and 0.56 ± 0.37 for area B (P = 0.25), 40.6 ± 8.0 and 15.8 ± 4.3 for area C (P = 0.02), and 41.3 ± 9.3 and 22.9 ± 5.6 for area D (P = 0.12).



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  		FIG. 3. Mean ± SEM percentage of activated (cFos-ir containing) GnRH-ir neurons in brain areas B–D of OVX+EB rats. The percentage of activated GnRH-ir neurons was significantly decreased in octreotide-treated (n = 8) rats compared to saline-treated rats (n = 9) (P = 0.042)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study clearly demonstrates that a single central injection with an SOM analog (octreotide) just prior to surge onset significantly decreased the E2-induced LH surge in OVX female rats. Moreover, 7 out of the 10 animals that were treated with octreotide showed no apparent LH surge at all. These findings are in agreement with both in vitro and in vivo studies showing that SOM is able to decrease LH secretion in male rats [12, 13]. However, in those studies, prolonged treatment was used to induce high levels of SOM, whereas we applied the SOM analog only once within the so-called critical period [23, 24]. Moreover, in previous studies, male rats were used, which do not normally express an LH surge. The present results suggest that SOM, apart from possible effects on basal LH release, may also affect episodic preovulatory LH release during the reproductive cycle in the female.

In female rats, both hypothalamic SOM peptide and pre-pro SOM mRNA levels fluctuate during the estrous cycle [25–27]. Hypothalamic pre-pro SOM mRNA levels [26] as well as SOM release from the median eminence (ME) [27] are maximal at proestrus afternoon, whereas SOM peptide levels in the preoptic area (POA), posterior hypothalamus, and ME are decreased on proestrus compared to all other days of the estrous cycle [25]. These data may suggest that in cycling female rats, SOM release from the hypothalamus peaks on proestrus afternoon. In addition, our present data clearly show that centrally applied SOM decreases LH release, suggesting that SOM may be involved in the regulation of the preovulatory LH surge in female rats.

We injected the SOM analog in the lateral ventricle of the brain, assuming that the inhibiting effect on LH release is caused by direct effects of SOM at the hypothalamic level, leading to changes in the activity of hypothalamic GnRH cells, or GnRH release from the ME, or both. Indeed, we found a significant decrease in the percentage of activated GnRH neurons (i.e., the neurons containing a cFos-positive nucleus) after octreotide treatment. We studied the activity of GnRH neurons during the ascending phase of the LH surge, because it is well known that at that phase, a positive correlation exists between the percentage of activated GnRH cells and plasma LH concentrations [19, 28–30]. We particularly evaluated the GnRH cells in the areas that are known to become activated during the preovulatory LH surge (i.e., the DBB, OVLT, and MPO) [19, 22, 30, 31]. The decrease in activated GnRH neurons following octreotide treatment was, interestingly, most prominent at the level of the OVLT, the region in which the first activation of GnRH neurons during LH surge onset occurs [19, 22, 31]. Our data strongly suggest that the decreased LH release we found after a single central injection with the SOM analog octreotide is, at least partially, the result of a direct effect on hypothalamic GnRH neuron activation, most likely resulting in a reduction in GnRH release, leading to the observed decrease in LH release from the pituitary.

On the day of proestrus, rising levels of E2 together with the feedback of progesterone lead to an increase in GnRH release, evoking the LH surge. In the rat, the descending phase of the LH surge is most likely the direct result of a decrease in GnRH release from the hypothalamus [32, 33]. We hypothesize that the increasing levels of hypothalamic SOM on proestrus afternoon may be involved in this process. In view that in the present study the LH surge was completely abolished in 7 out of 10 animals after octreotide administration in the "critical period," we suggest that SOM may be involved in the descending rather than the ascending phase of the LH surge. However, the precise physiological role and mechanism of action of SOM in the regulation of the preovulatory LH surge remains to be determined.

Apart from a decrease in LH release, we also found a decrease in GH release from the pituitary within 2.5 h after the central administration of octreotide. Physiological regulation of GH release involves, besides direct projections of periventricular SOM cells to the ME [34–38], projections of hypothalamic SOM cells to the GHRH cells in the ARC. Via these projections, SOM can decrease GHRH release from the ME, leading to a decrease in GH release from the pituitary [39–44]. It is also possible that the octreotide we injected into the lateral ventricle may have affected the GHRH cells located very close to the third ventricle in the ARC [45], resulting in a decreased GH release. However, we cannot exclude the possibility that the centrally injected SOM analog also reached the pituitary by passing the blood-brain barrier (BBB), and directly affected both pituitary gonadotrophs and somatotrophs via the portal vessel system. It has been shown that several SOM analogs can pass the BBB by a saturable transport mechanism [46]. Moreover, several in vivo studies have shown that SOM may directly affect the gonadotropic cells in the pituitary [14–16]. Anterior pituitary cells express all five SOM receptors (sstr1–5), but LH cells predominantly express sstr2, whereas GH cells mainly express sstr4 and sstr5 [47]. E2 is able to upregulate sstr2 and sstr3 [48–50], suggesting that these SOM receptors on the pituitary LH cells may be up-regulated by the increasing E2 levels on the day of proestrus. Octreotide has a high binding affinity for both sstr2 and sstr5 and moderate for sstr3, but has only a low binding affinity for sstr1 and sstr4 [50, 51]. Consequently, a direct effect of octreotide on LH and GH release from the pituitary cannot be excluded in the present experimental setup. In addition, several studies showed that a subset of somatotrophs is multipotential and produces not only GH, but also LH and FSH mRNA. Moreover, these cells express both GHRH and GnRH receptors [52–55], indicating that effects at the level of the pituitary may also have played an additional role. However, that we found an effect of centrally injected octreotide at the level of the hypothalamus (i.e., a significant decrease in activated GnRH cells), supports the idea that octreotide, at least partially, may have also affected the control of LH and GH release at the level of the hypothalamus.

In the hypothalamus, expression of sstr1 and sstr2 is high, expression of sstr3 is moderate, and sstr4 and sstr5 expression is very low [56]. SOM cells are widely distributed in the rat brain [51], but only SOM cells in the rostral part of the PeVN are responsible for SOM release from the ME, which is involved in the regulation of GH release from the pituitary [34, 36–38]. In PeVN, sstr1 and sstr2 mRNA, which are colocalized with SOM cells, have been demonstrated [51, 57, 58], whereas in the ARC, sstr1–4 have been found [51, 57, 59–61]. GHRH neurons were found to be predominantly coexpressed with sstr1 and sstr2, hardly with sstr3 and sstr4, and not with sstr5 [58, 62]. Also, in the hypothalamic areas that contain GnRH neurons, sstr expression has been demonstrated as follows: sstr1–3 expression has been found in the anterior hypothalamus [59, 63], sstr1,2 in the MPO [51, 63], and sstr1 in the OVLT [57]. Because octreotide shows highest affinity for sstr2, sstr3, and sstr5, which are found in or near areas that contain GnRH and GHRH neurons, the changes in LH and GH release as found in the present study could very well be the result of a direct action of this SOM analog at the level of the hypothalamus leading to changes in the activity of GnRH and GHRH neurons. The hypothalamic circuitry involved in these effects, however, remains to be established.

To our knowledge, no published data exist on possible direct innervation of GnRH by SOM cells, nor on the presence of specific SOM receptors on GnRH cells in the OVLT, MPO, or both. Although studies in the ewe provided evidence for a direct input from the ventromedial nucleus (VMN) to GnRH neurons [64], in the rat, fibers that arose from the VMN were found to project to the periventricular POA (pePOA) [65]. The pePOA itself does not contain GnRH neurons [21, 29], but was found to contain neurons that become activated concurrent with the activation of GnRH neurons surrounding the OVLT at the time of the preovulatory LH surge and project to these GnRH neurons at this time [22, 29, 65–67]. Possibly, the neurons projecting from the pePOA to GnRH cells at the time of the preovulatory LH surge are {gamma}-aminobutyric acid-, or neurotensin-, or glutamate-containing neurons, or a combination of these, because these neurons are located in the POA and were found to innervate GnRH neurons in the OVLT and POA [68]. Thus, if SOM is involved in the regulation of the LH surge by affecting the activity of GnRH cells, its effect in the female rat is more likely to be indirect. Because sstr1 is located in both the POA [51] and in the OVLT [57], SOM may very well directly affect the activity of neurons in these areas. Indeed, lesions of the anterior hypothalamic area (AHA) resulted in reduced SOM levels in the POA [34], suggesting that SOM cells from the AHA (i.e., the hypothalamic region that contains SOM cells projecting to the ME to regulate GH release) also project to the POA to send signals to GnRH cells in this region.

In conclusion, a single i.c.v. injection of a somatostatin analog during the so-called critical period resulted in a significant attenuation of the E2-induced LH surge in OVX rats. Our results strongly suggest that this was the direct result of, at least partially, a significant inhibition of the activation of GnRH cells in the hypothalamus. These results, together with the observed changes in hypothalamic SOM activity and release over the estrous cycle, as previously described in the literature, led us to propose the hypothesis that SOM may be involved in the normal physiological regulation of the descending phase of the proestrus LH surge.


   ACKNOWLEDGMENTS
 
We thank Jan Kastelijn (Animal Sciences Group, Wageningen University, Wageningen) for his practical assistance.


   FOOTNOTES
 
1 This work was supported by the Wageningen Institute of Animal Sciences, Graduate School of Wageningen University. Back

2 Correspondence: Harmke H. Van Vugt, Human and Animal Physiology Group, Animal Sciences Group, Wageningen University, Haarweg 10, 6709 PJ Wageningen, The Netherlands. FAX: 31 317 484077; harmke.vanvugt{at}wur.nl Back

Received: 9 March 2004.

First decision: 28 March 2004.

Accepted: 6 May 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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