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BOR - Papers in Press, published online ahead of print October 23, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.005959
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BIOLOGY OF REPRODUCTION 68, 363–369 (2003)
DOI: 10.1095/biolreprod.102.005959
© 2003 by the Society for the Study of Reproduction, Inc.

Pituitary

Testosterone-Dependent Effects of Galanin on Pituitary Luteinizing Hormone Secretion in Male Rats1

Joseph R. Scheffena, Cynthia L. Spletta, Joshua A. Desotellea, and Angela C. Bauer-Dantoin2,a

a Department of Human Biology, University of Wisconsin-Green Bay, Green Bay, Wisconsin 54311-7001


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Galanin is a 29-amino-acid peptide that colocalizes with GnRH in hypothalamic neurons. High concentrations of galanin are present in portal vessel blood of both male and female rats, and galanin receptors are present on gonadotropes in both sexes. Results from studies of female rats indicate that galanin acts at the level of the pituitary to directly stimulate LH secretion and also to enhance GnRH-stimulated LH secretion. The effects of galanin on pituitary LH secretion in male rats are relatively uncharacterized; thus, the present in vivo study was conducted 1) to examine the ability of galanin to affect basal or GnRH-stimulated LH secretion in male rats and 2) to determine whether the effects of galanin on LH secretion in male rats are testosterone-dependent. All three doses of galanin used (1, 5, and 10 µg/pulse) significantly enhanced GnRH-stimulated LH secretion in intact male rats. Only the highest dose of galanin directly stimulated LH secretion (without GnRH coadministration) in intact males. Galanin did not directly stimulate LH secretion or enhance GnRH-stimulated LH secretion in castrated male rats. In fact, the highest dose of galanin inhibited GnRH-stimulated LH secretion in castrated males. Upon testosterone replacement, the ability of galanin to directly stimulate LH secretion and to enhance GnRH-stimulated LH secretion was restored in castrated males. These results suggest a role for galanin in the regulation of LH release in male rats and demonstrate that testosterone upregulates the ability of the pituitary to respond to the stimulatory effects of galanin.

galanin, gonadotropin-releasing hormone, luteinizing hormone, male reproductive tract, pituitary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Galanin is a 29-amino-acid peptide that was originally isolated from porcine intestine [1] and is widely expressed throughout the central and peripheral nervous systems of a number of species [2–4]. High concentrations of galanin are present in the hypothalamus, where it plays a role in the regulation of a number of physiological processes in mammals, including food intake, body weight maintenance, growth, and reproduction [5, 6].

Hypothalamic galanin neurons appear to regulate reproductive function at two distinct levels within the reproductive axis. The first level is within the hypothalamus itself, where galanin stimulates GnRH neurosecretion in both male and female rats [7–9]. It has been hypothesized that the hypothalamic effects of galanin on GnRH secretion are important for two critical reproductive events: induction of GnRH neuronal activity at the onset of puberty in both males and females and generation of the preovulatory LH surge in females [10]. Evidence in support of the puberty hypothesis comes from studies documenting a significant increase in hypothalamic galanin gene expression at the onset of puberty in both sexes [11]. A role for the peptide in the generation of preovulatory LH surges has been suggested by the observation that estrogen induces galanin gene expression in hypothalamic neurons [12, 13] and by in vivo studies demonstrating that intracerebroventricular administration of the galanin receptor-antagonist galantide suppresses the proestrous LH surge in female rats (presumably by interfering with galanin's ability to participate in generation of the GnRH surge) [9].

Hypothalamic galanin neurons also act as hypophysiotropic regulators of the reproductive axis. The role of hypothalamic galanin as a regulator of pituitary reproductive hormone secretion was first hypothesized when immunocytochemical studies demonstrated the presence of galanin nerve terminals in the median eminence [14, 15]. It was found that in both sexes, a subset of these neurons coexpresses both galanin and GnRH [16, 17], and portal blood measurements of galanin and GnRH have demonstrated that the two peptides are cosecreted in a pulsatile manner into the portal vasculature of female rats [18]. Subsequent in vitro studies with female pituitary cell cultures demonstrated that the primary pituitary effect of galanin is to enhance GnRH-stimulated LH secretion, although pharmacologic doses of the peptide can directly stimulate LH secretion [18]. The pituitary effects of galanin, like the hypothalamic effects, appear to be important for the generation of preovulatory LH surges in females, because peripheral administration of the galanin receptor-antagonist galantide significantly reduces the amplitude of LH surges in steroid-primed, ovariectomized rats [9].

The effects of galanin on pituitary LH secretion in males remain largely uncharacterized. The vast majority of the studies regarding the reproductive effects of galanin have been conducted in female rats, because the neurons coexpressing galanin and GnRH are more abundant in the female hypothalamus than in the male hypothalamus [19]. However, evidence suggests that the peptide plays a role in regulating pituitary LH secretion in males as well. This evidence includes the findings that galanin is secreted into the portal vasculature of male rats at high concentrations and that galanin receptors are present on gonadotropes within the male pituitary [20]. Therefore, in the present study, we hypothesized that galanin plays a direct pituitary role in regulating LH secretion in male rats, either by stimulating LH secretion on its own or by enhancing GnRH-stimulated LH secretion. Additionally, we were interested in determining whether the pituitary effects of galanin on LH secretion in males are testosterone (T)-dependent, because a galanin-receptor subtype known to be expressed in gonadotropes (GalR2) [20] is influenced by the presence of T in hypothalamic neurons [21]. Thus, the present study was conducted to characterize the pituitary effects of galanin (with or without GnRH coadministration) on LH secretion in intact male, castrated male, and castrated, T-replaced male rats.


   MATERALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Surgical Protocol

All surgical and experimental procedures were conducted in accordance with the policies of the University of Wisconsin-Green Bay's Animal Care and Use Committee. Male Sprague-Dawley rats (weight, 200–250 g; Charles River Laboratories, Wilmington, MA) were housed in groups of two to three rats per cage in a temperature- and humidity-controlled room, with lights-on from 0600 to 2000 h. Animals had access to tap water and standard laboratory rat chow ad libitum.

The pituitary effects of galanin were examined in three groups of animals: 1) intact (sham-castrated) male rats, 2) castrated male rats, and 3) castrated, T-replaced male rats. Eight days before experiments, rats were anesthetized with halothane (Halocarbon Laboratories, Hackensack, NJ) and either castrated or sham-castrated. Sham-castrated rats were implanted with s.c. empty Silastic capsules (length, 30 mm; inner diameter, 0.062 inches; outer diameter, 0.125 inches; Dow Corning, Midland, MI). Castrated rats were implanted with either empty Silastic capsules or Silastic capsules filled with crystalline T (Sigma-Aldrich Co., St. Louis, MO).

On the day before experiments, rats were again anesthetized with halothane and fitted with indwelling jugular catheters (PE50). The catheter was inserted into the atrium, tunneled s.c. to the nape, and exteriorized by means of a small plastic cuff. A stainless-steel plug was used to occlude the catheter until experiments were conducted on the following day.

Pulsatile Hormone Injections and Blood Sampling

From 1100 to 1800 h on the day of experiments, hourly blood samples were withdrawn from the atrial catheter and centrifuged, and the plasma was stored at -20°C (n = 4–11 animals/treatment group). Blood samples were 0.27 ml in volume and were replaced with an equal volume of heparinized saline. At 1230 h, rats received an i.p. injection of pentobarbital (40 mg/kg body weight; Sigma-Aldrich Co.) to block hypothalamic GnRH neurosecretion [22]. Control rats received i.p. injections of an equal volume of 0.9% saline. At 30-min intervals between 1300 and 1700 h, pentobarbital-blocked rats received i.v. injections of either saline, GnRH alone (25 ng/pulse; Sigma-Aldrich Co.), galanin alone (1, 5, or 10 µg/pulse; rat galanin; Peninsula Laboratories, Belmont, CA), or galanin (1, 5, or 10 µg/pulse) plus GnRH (25 ng/pulse). The three doses of galanin were chosen to produce peak peripheral blood concentrations that were, respectively, equal to, 5-fold greater than, or 10-fold greater than peak concentrations observed in portal vessel blood [18]. Likewise, the dose of GnRH was chosen to produce a peripheral concentration of the decapeptide that was equivalent to concentrations observed in portal vessel blood at the peak of a GnRH pulse [18]. Both GnRH and galanin were administered in a pulsatile manner, rather than as a continuous infusion, to mimic the endogenous secretory profiles of the peptides. The combined galanin/GnRH infusions were carried out by injection of an appropriate mixture of the two peptides in a single solution. The injection volume in all groups was 100 µl.

Radioimmunoassays

Levels of LH in plasma samples were determined using RIAs performed by the Radioimmunoassay Laboratory at the University of Wisconsin School of Veterinary Medicine (Madison, WI). Assay materials were generously provided by the NIDDK. The standard used in the assays was LH RP-3. The intra- and interassay coefficients of variation were 6% and 8%, respectively. The sensitivity of the LH assay was 0.28 ng/ml.

Levels of T in plasma samples were measured using a single RIA performed by the Assay Services Unit of the Wisconsin Regional Primate Research Center at the University of Wisconsin-Madison. The intraassay coefficient of variation was 12%.

Statistical Analysis of Data

Plasma LH levels in each animal were calculated as the average amount of LH released from 1400 to 1700 h. The 1800-h LH measurements were not included in the data analyses, because observations of LH levels and behavioral activity in pentobarbital-treated control rats indicated that the pentobarbital effects were beginning to wear off at this time. Group means were generated, and comparisons between mean LH levels in response to saline treatment vs. hormone treatments (galanin alone, GnRH alone, or GnRH + galanin) within a steroid treatment group (intact male, castrated male, or castrated, T-replaced male rats) were made using the Student t-test. In all cases, differences were considered to be significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Plasma T Levels in Steroid Treatment Groups

Mean plasma T levels in intact rats were 3.61 ± 0.52 pg/ml (n = 5 in each group unless noted otherwise; this and subsequent values are expressed as mean ± SEM). Castration of male rats resulted in a significant reduction in plasma T levels (to 0.62 ± 0.13 pg/ml; P < 0.001). Steroid replacement of castrated rats with s.c. T implants produced a mean plasma T level of 2.95 ± 0.21 pg/ml, which was comparable to the mean T level observed in the intact rats.

Effects of Pentobarbital on LH Release

Castrated rats treated with an i.p. injection of saline at 1230 h and pulses of saline from 1300 to 1700 h exhibited a mean plasma LH level of 3.06 ± 0.18 ng/ml between 1400 and 1700 h (Fig. 1). Administration of pentobarbital at 1230 h to castrated, saline-treated rats resulted in a significant decline in mean plasma LH levels (2.16 ± 0.30 ng/ml; P < 0.05). This decline in LH levels resulted from the barbiturate's inhibitory influence on hypothalamic GnRH neurosecretion [22].



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  		FIG. 1. Effects of i.p. administration of saline or pentobarbital (40 mg/kg body weight) at 1230 h on LH levels (mean ± SEM) between 1400 and 1700 h in 1) castrated male rats, 2) intact male rats, and 3) castrated, T-replaced male rats. All animals received pulsatile, saline-only infusions every 30 min from 1300 to 1700 h. *P < 0.05 vs. rats in the same steroid treatment group that were treated with an i.p. injection of saline at 1230 h

Pentobarbital treatment of intact rats that received saline-only pulses from 1300 to 1700 h resulted in a mean plasma LH level of 0.42 ± 0.05 ng/ml between 1400 and 1700 h (n = 4). This mean was not significantly different from that observed in intact control rats treated with an i.p. injection of saline at 1230 h (0.43 ± 0.05 ng/ml; n = 4). Likewise, pentobarbital treatment of castrated, T-replaced control rats resulted in a mean plasma LH level of 0.28 ± 0.003 ng/ml from 1400 to 1700 h (n = 7), which was not significantly different from that observed in castrated, T-replaced rats treated with an i.p. injection of saline at 1230 h (0.301 ± 0.02 ng/ml; n = 6). Thus, the effects of pentobarbital blockade of GnRH neurosecretion on pituitary LH secretion were not evident in intact or castrated, T-replaced control rats. This can be attributed to the presence of T negative feedback in these groups of animals, the mean plasma LH levels of which (even in the absence of pentobarbital blockade) were at or near the level of detectability in the LH RIA.

Effects of Galanin Alone on LH Secretion in Intact Male Rats

Of the three doses of galanin used in the present study (1, 5, or 10 µg/pulse), only a dose of 10 µg/pulse given every 30 min from 1300 to 1700 h caused a significant elevation in mean plasma LH levels between 1400 and 1700 h in intact male rats (0.97 ± 0.11 ng/ml; P < 0.005) (Fig. 2). Administration of galanin at a dose of 1 µg/pulse resulted in a mean LH level of 0.45 ± 0.09 ng/ml between 1400 and 1700 h in the intact rats, whereas administration of the hormone at a dose of 5 µg/pulse resulted in a mean LH level of 0.46 ± 0.11 ng/ml in the intact rats. Neither of these levels differed significantly from mean LH levels observed in saline-treated, pentobarbital-blocked intact rats.



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  		FIG. 2. Effects of pulsatile galanin administration on LH levels (mean ± SEM) between 1400 and 1700 h in pentobarbital-blocked, intact male rats. Pulses of galanin (or saline) were administered every 30 min from 1300 to 1700 h. *P < 0.005 vs. saline-treated controls

Effects of Galanin on GnRH-Stimulated LH Secretion in Intact Male Rats

The effects of galanin on GnRH-stimulated LH secretion are illustrated in Figure 3. Pulsatile administration of GnRH alone at a dose of 25 ng/pulse from 1300 to 1700 h resulted in a mean plasma LH level of 1.06 ± 0.07 ng/ml between 1400 to 1700 h in intact rats (n = 6; P < 0.005 vs. saline-treated controls). Simultaneous administration of 1 µg/pulse of galanin along with the same dose of GnRH resulted in a mean plasma LH level of 1.67 ± 0.29 ng/ml in the intact rats, which was 67% greater than the mean LH level observed after pulsatile administration of GnRH alone (P < 0.05). Likewise, coadministration of galanin at either 5 or 10 µg/pulse along with GnRH resulted in mean plasma LH levels between 1400 to 1700 h (1.60 ± 0.10 and 1.69 ± 0.14 ng/ml, respectively) that were significantly greater than those levels observed in the intact rats treated with GnRH alone (P < 0.005 in both cases).



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  		FIG. 3. Effects of galanin on GnRH-stimulated LH secretion in pentobarbital-blocked, intact male rats. Hormone (or saline) pulses were administered every 30 min from 1300 to 1700 h. Values represent LH levels (mean ± SEM) between 1400 and 1700 h. *P < 0.005 vs. saline-treated controls, **P < 0.05 vs. intact animals treated with GnRH alone

Effects of Galanin Alone on LH Secretion in the Absence of Gonadal T

Mean LH levels observed in castrated male rats treated with 1 or 5 µg/pulse of galanin were 2.31 ± 0.46 and 2.97 ± 0.33 ng/ml, respectively (Fig. 4). These LH levels did not differ significantly from those observed in pentobarbital-blocked, saline-treated castrated male rats. Pulsatile administration of galanin at a dose of 10 µg/pulse between 1300 and 1700 h resulted in a mean plasma LH level of 1.62 ± 0.20 ng/ml between 1400 and 1700 h, which was significantly lower than the level observed in pentobarbital-blocked, saline-treated castrated rats (P < 0.05).



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  		FIG. 4. Effects of pulsatile galanin administration on LH levels (mean ± SEM) between 1400 and 1700 h in pentobarbital-blocked, castrated male rats. Pulses of galanin (or saline) were administered every 30 min from 1300 to 1700 h. *P < 0.05 vs. saline-treated controls

Effects of Galanin on GnRH-Stimulated LH Secretion in the Absence of Gonadal T

Figure 5 depicts the effects of galanin on GnRH-stimulated LH secretion in castrated male rats. Galanin was found to have an inhibitory effect on GnRH-stimulated LH secretion in the absence of gonadal T; this effect was only observed on coadministration of the highest dose of galanin (10 µg/pulse) with GnRH to castrated males. Pulsatile administration of 25 ng/pulse of GnRH alone resulted in a mean plasma LH level of 5.36 ± 0.59 ng/ml (n = 6). Coadministration of 1 or 5 µg/pulse of galanin along with the same dose of GnRH produced mean LH levels of 4.09 ± 0.60 ng/ml (n = 4) and of 4.16 ± 0.41 ng/ml (n = 6), respectively. These means were not significantly different from those produced by administration of GnRH alone. However, when 10 µg/pulse of galanin was coadministered with GnRH from 1300 to 1700 h to castrated males, a mean LH level of 3.60 ± 0.47 ng/ml resulted. This mean was significantly lower than that observed upon pulsatile administration of GnRH alone (P < 0.05).



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  		FIG. 5. Effects of galanin on GnRH-stimulated LH secretion in pentobarbital-blocked, castrated male rats. Hormone (or saline) pulses were administered every 30 min from 1300 to 1700 h. Values represent LH levels (mean ± SEM) between 1400 and 1700 h. *P < 0.001 vs. saline-treated, castrated controls; **P < 0.05 vs. castrated animals treated with GnRH alone

Effects of Galanin on Basal and GnRH-Stimulated LH Secretion in Castrated, T-Replaced Male Rats

A final series of experiments was conducted in castrated, T-replaced male rats to determine whether T is the requisite component of the hormonal milieu in gonadally intact male rats that allows the stimulatory effects of galanin on basal and GnRH-stimulated LH secretion to be expressed. The dose of galanin that was found both to directly stimulate LH secretion and to enhance GnRH-stimulated LH secretion in intact rats (10 µg/pulse) was chosen for the study.

As illustrated in Figure 6, castrated, T-replaced male rats exhibited a mean LH level of 0.79 ± 0.05 ng/ml (n = 11) between 1400 and 1700 h in response to administration of 25 ng/pulse of GnRH. Coadministration of 10 µg/pulse of galanin along with the same dose of GnRH resulted in a mean LH level of 1.19 ± 0.2 ng/pulse between 1400 and 1700 h, which was significantly greater than the mean level observed on pulsatile administration of GnRH alone (P < 0.01). Administration of 10 µg/pulse of galanin alone between 1300 and 1700 h resulted in a mean LH level of 0.61 ± 0.12 ng/ml in castrated, T-replaced males, which was 2-fold greater than the mean LH level observed in pentobarbital-blocked, saline-treated rats in the same steroid treatment group (P < 0.01) (Fig. 6).



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  		FIG. 6. Effects of galanin on basal and GnRH-stimulated LH secretion in castrated, T-replaced male rats. Hormone (or saline) pulses were administered every 30 min from 1300 to 1700 h. Values represent LH levels (mean ± SEM) between 1400 and 1700 h. *P < 0.001 vs. saline-treated controls, **P < 0.01 vs. animals treated with GnRH alone


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To our knowledge, the present study represents the first in vivo demonstration that galanin plays a direct pituitary role in regulating LH secretion in male rats. Galanin administration was found to significantly augment pituitary responsiveness to the stimulatory actions of GnRH in intact male rats. Galanin was also found to stimulate pituitary LH secretion on its own—without GnRH coadministration—in intact males. However, unlike the potent effects of galanin on GnRH-stimulated LH secretion, the stimulatory effects of galanin alone on basal LH secretion were observed only after pulsatile administration of a supraphysiological dose of the peptide, suggesting that the primary pituitary effect of galanin in intact males is to enhance the actions of GnRH. These observations, taken together with the findings that galanin is present at high concentrations in the portal vasculature of male rats [18] and that gonadotropes in the male pituitary express galanin receptors [20], provide strong evidence in support of the hypothesis that galanin acts as a hypophysiotropic regulator of pulsatile LH secretion in male rats.

Of note in the present study is the observation that the stimulatory effects of galanin on basal and GnRH-stimulated LH secretion occur only in the presence of physiological levels of circulating T. The stimulatory effects of galanin were completely absent in castrated male rats that received no further T replacement. These findings indicate that the ability of the pituitary to respond to the stimulatory effects of galanin is dependent on the presence of gonadal steroids in males. It remains to be determined how androgens enhance pituitary responsiveness to the stimulatory actions of galanin. One possible explanation is that androgens upregulate the expression of a specific galanin-receptor subtype that is positively coupled to LH secretion in pituitary gonadotropes. A likely candidate for such a receptor is the GalR2 receptor, because in situ hybridization studies have demonstrated the presence of significant quantities of the mRNA that encodes this specific receptor subtype in the pituitary gonadotropes of intact males [20]. Interestingly, expression of the GalR2 receptor in hypothalamic neurons is influenced by the presence of T; castration of male rats results in a significant decline in the labeling density of GalR2-receptor mRNAs in the arcuate nucleus of male rats [21]. Thus, GalR2 mRNA expression in the pituitary gland may also be T-dependent, with castration causing a decline in the expression of the receptor that, in turn, results in the inability of pituitary gonadotropes to respond to the stimulatory actions of galanin. Additional studies will be necessary to test this hypothesis and to examine whether the effects of T observed in the present study are exerted via androgen receptors or whether the steroid is acting via estrogen receptors after aromatization, as is the case for other effects of T within gonadotropes [23]

Not only were the stimulatory effects of galanin absent in castrated male rats of the present study, galanin was actually found to have an inhibitory effect on both basal and GnRH-stimulated LH secretion in the absence of gonadal T. The physiological relevance of these inhibitory effects of galanin remains to be determined, because they were observed only on administration of a supraphysiological dose of the peptide. However, it is interesting to note that our laboratory and others have observed similar inhibitory effects of galanin on pituitary LH secretion in ovariectomized female rats (unpublished data) and in female pituitary glands cultured in the absence of gonadal steroids [24]. Perhaps in the absence of gonadal steroids, the expression of an inhibitory galanin-receptor subtype—such as the GalR1-receptor subtype, which is known to couple with the Gi protein [25]—predominates, and activation of the receptor by galanin results in an inhibition of second-messenger systems known to mediate GnRH-stimulated LH secretion.

It can be concluded that the primary pituitary effect of galanin in intact male rats is not to directly stimulate LH secretion on its own but, rather, to enhance GnRH-stimulated LH secretion. This conclusion is based on the findings in the present study that all three doses of galanin examined were capable of enhancing GnRH-stimulated LH secretion in intact males, whereas only the highest dose of galanin was capable of stimulating LH secretion on its own without GnRH coadministration. Thus, it seems that the effects of galanin on pituitary LH secretion in males are primarily modulatory in nature. The ability of galanin to modulate GnRH-stimulated LH secretion was observed at a physiologically relevant dose in the present study: administration of the lowest dose of galanin (1 µg/pulse) was estimated to produce peripheral plasma concentrations of the peptide that are equivalent to portal vessel blood concentrations of the peptide observed at the peak of galanin pulses in females and that are 10-fold greater than basal portal blood concentrations measured in male rats [18]. The significance of the rise in LH levels in intact males in response to administration of the supraphysiological dose of galanin (10 µg/pulse) alone remains to be determined. Given that galanin is capable of stimulating hypothalamic GnRH neurosecretion [7–9], it is plausible that peripheral infusions of high doses of the peptide override pentobarbital blockade of GnRH neurosecretion, stimulate its release (or that of another hypothalamic factor) from nerve terminals in the median eminence, and thereby indirectly cause an increase in pituitary LH secretion. Thus, additional in vitro studies will be necessary to confirm a direct, stimulatory effect of galanin alone on pituitary LH secretion.

Interestingly, the stimulatory effects of galanin on GnRH-stimulated LH secretion in intact male rats were not found to be dose-dependent in the present study. A possible explanation for this lack of dose-dependence may be that upon coadministration of the lowest dose of galanin (1 µg/pulse) along with 25 ng/pulse of GnRH, gonadotropes of intact males are responding maximally to stimulation by the neuropeptides and, thus, administration of higher concentrations of galanin produces no further increase in LH secretion. Indeed, mean LH levels observed in the GnRH with 1 µg/pulse of galanin treatment group were 4-fold greater than levels observed in saline-treated controls.

As mentioned previously, combined in situ hybridization immunohistochemistry studies have demonstrated the presence of high levels of GalR2-receptor mRNAs in gonadotropes of the intact male pituitary gland [20]. Pharmacological studies of the GalR2 receptor have demonstrated that the receptor is capable of stimulating inositol phospholipid turnover, mobilizing intracellular calcium [25, 26] and stimulating MAP kinase activity [25]. Given that these intracellular signaling molecules are also known to mediate GnRH-stimulated LH secretion in pituitary gonadotropes [27–29], it appears likely that galanin modulation of GnRH-stimulated LH secretion takes place, at least in part, at the level of intracellular signal transduction mechanisms. The possibility also exists that galanin modulates GnRH-receptor expression in the male pituitary, given that Parker et al. [30] have demonstrated a modest stimulatory effect of peptide on GnRH-agonist binding to female anterior pituitary membranes. Further studies will be required to investigate these hypotheses.

In conclusion, to our knowledge the present study provides the first direct, in vivo demonstration of a pituitary role for galanin in regulating LH secretion in male rats. The primary pituitary effect of galanin observed in the present study was to enhance GnRH-stimulated LH secretion. This modulatory effect of galanin only occurred in the presence of physiological levels of T; galanin did not enhance GnRH-stimulated LH secretion in castrated male rats that received no further T replacement. These results, taken together with the findings that galanin is secreted into the portal vasculature of male rats at high concentrations [18] and that galanin receptors are present on gonadotropes within the male pituitary [20], indicate a role for the neuropeptide in the generation of pulsatile LH release in male rats. Further studies likely will delineate whether the pituitary actions of galanin play a role in regulating important reproductive events in the male, such as activation of the reproductive axis at the time of puberty.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Craig Hanke for his critical review of the manuscript.


   FOOTNOTES
 
1 Supported by NIH Academic Research Enhancement Award R15-HD37951-01 (to A.B.D.). Back

2 Correspondence: Angela Bauer-Dantoin, University of Wisconsin-Green Bay, Department of Human Biology, Environmental Sciences Building, Room 301, 2420 Nicolet Drive, Green Bay, WI 54311-7001. FAX: 920 465 2769: bauera{at}uwgb.edu Back

Received: 25 March 2002.

First decision: 10 April 2002.

Accepted: 7 August 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Tatemoto K, Rokaeus A, Hornvall J, McDonald TJ, Mutt V. Galanin—a novel biologically active peptide from porcine intestine. FEBS Lett 1983 164:124-128[CrossRef][Medline]
  2. Rokaeus A. Galanin: a newly isolated biologically active neuropeptide. Trends Neurosci 1987 10:158-164
  3. Skofitsch G, Jacobowitz DM. Immunohistochemical mapping of galanin-like neurons in the rat central nervous system. Peptides 1985 6:509-546[CrossRef][Medline]
  4. Skofitsch G, Jacobowitz DM. Quantitative distribution of galanin-like immunoreactivity in the central nervous system. Peptides 1985 7:609-613
  5. Merchenthaler I, Lopez FJ, Negro-Vilar A. The physiology of galanin-containing pathways in the central nervous system. Prog Neurobiol 1993 40:711-769[CrossRef][Medline]
  6. Kaplan LM, Hooi SC, Arabczinskas DR, Strauss RM, Davidson MB, Hsu DW, Koenig JI. Neuroendocrine regulation of galanin gene expression. In: Hokfelt T, Bartfai T, Jacobowitz D, Ottoson D (eds.), Galanin: A Multifunctional Peptide in the Neuro-endocrine System. New York: Macmillan Press; 1990: 43–65
  7. Sahu A, Crowley WR, Tatemoto K, Balasubramaniam A, Kalra SP. Effects of neuropeptide Y (NPY), NPY analog (norleucine-NPY), galanin and neuropeptide K on LH release in ovariectomized (OVX) and OVX, estrogen, progesterone treated rats. Peptides 1987 8:921-926[CrossRef][Medline]
  8. Lopez FJ, Negro-Vilar A. Galanin stimulates luteinizing hormone-releasing hormone secretion from arcuate nucleus–median eminence fragments in vitro: involvement of an {alpha}-adrenergic mechanism. Endocrinology 1990 127:2431-2436[Abstract/Free Full Text]
  9. Sahu A, Xu B, Kalra SP. Role of galanin in stimulation of pituitary luteinizing hormone secretion as revealed by a specific receptor antagonist, galantide. Endocrinology 1994 134:529-536[Abstract/Free Full Text]
  10. Rossmanith WG, Clifton DK, Steiner RA. Galanin gene expression in hypothalamic GnRH-containing neurons of the rat: a model for autocrine regulation. Horm Metab Res 1996 28:257-266[Medline]
  11. Rossmanith WG, Marks DL, Clifton DK, Steiner RA. Induction of galanin gene expression with onset of puberty in the rat: its dependence on sex steroids. Endocrinology 1994 135:1401-1408[Abstract]
  12. Marks DL, Lent KL, Rossmanith WG, Clifton DK, Steiner RA. Activation-dependent induction of galanin mRNA expression in GnRH neurons in the rat. Endocrinology 1994 134:1991-1998[Abstract]
  13. Rossmanith WG, Marks DL, Clifton DK, Steiner RA. Induction of galanin mRNA in GnRH neurons by estradiol and its facilitation by progesterone. J Neuroendocrinol 1996 8:185-191[CrossRef][Medline]
  14. Merchenthaler I. The hypophysiotropic galanin system of the rat brain. Neuroscience 1991 44:643-668[CrossRef][Medline]
  15. Arai R, Calas A. Ultrastructural localization of galanin immunoreactivity in the rat median eminence. Brain Res 1991 562:339-343[CrossRef][Medline]
  16. Merchenthaler I, Lopez FJ, Negro-Vilar A. Colocalization of galanin and luteinizing hormone-releasing hormone in a subset of preoptic hypothalamic neurons: anatomical and functional correlates. Proc Natl Acad Sci U S A 1990 87:6326-6330[Abstract/Free Full Text]
  17. Coen CW, Montagnese C, Opacka JJ. Coexistence of gonadotropin-releasing hormone and galanin: immunohistochemical and functional studies. J Neuroendocrinol 1990 2:107-111
  18. Lopez FJ, Merchenthaler I, Ching M, Wisniewski MG, Negro-Vilar A. Galanin: a hypothalamic-hypophysiotropic hormone modulating reproductive functions. Proc Natl Acad Sci U S A 1991 88:4508-4512[Abstract/Free Full Text]
  19. Liposits Z, Reid JJ, Negro-Vilar A, Merchenthaler I. Sexual dimorphism in copackaging of luteinizing hormone-releasing hormone and galanin into neurosecretory vesicles of hypophysiotropic neurons: estrogen dependency. Endocrinology 1995 136:1987-1992[Abstract]
  20. Depczynski B, Nichol K, Fathi Z, Iismaa T, Shine J, Cunningham A. Distribution and characterization of the cell types expressing GALR2 mRNA in brain and pituitary gland. Ann N Y Acad Sci 1998 863:120-128[CrossRef][Medline]
  21. Bouret S, Prevot V, Croix D, Howard A, Habert-Ortoli E, Jegou S, Vaudry H, Beauvillain J-C, Mitchell V. Expression of GalR1 and GalR2 galanin receptor messenger RNA in POMC neurons of the rat arcuate nucleus: effect of testosterone. Endocrinology 2000 141:1780-1794[Abstract/Free Full Text]
  22. Ching M. Correlative surges of LHRH, LH and FSH in pituitary stalk plasma and systemic plasma of rat during proestrus. Neuroendocrinology 1982 34:279-285[Medline]
  23. Lindzey J, Wetsel WC, Couse JF, Stoker T, Cooper R, Korach KS. Effects of castration and chronic steroid treatments on hypothalamic gonadotropin-releasing hormone content and pituitary gonadotropins in male wild-type and estrogen receptor-{alpha} knockout mice. Endocrinology 1998 139:4092-4101[Abstract/Free Full Text]
  24. Todd JF, Small CJ, Akinsanya KO, Stanley SA, Smith DM, Bloom SR. Galanin is a paracrine inhibitor of gonadotroph function in the female rat. Endocrinology 1998 139:4222-4229[Abstract/Free Full Text]
  25. Wang S, Hashemi T, Fried S, Clemmons AL, Hawes BE. Differential intracellular signaling of the GalR1 and GalR2 galanin receptor subtypes. Biochemistry 1998 37:6711-6717[CrossRef][Medline]
  26. Smith KE, Forray C, Walker MW, Jones KA, Tamm JA, Bard J, Branchek TA, Linemeyer DL, Gerald C. Expression cloning of a rat hypothalamic galanin receptor coupled to phosphoinositide turnover. J Biol Chem 1997 272:24612-24616[Abstract/Free Full Text]
  27. Andrews WV, Conn PM. Gonadotropin-releasing hormone stimulates mass changes in phosphoinositides and diacylglycerol accumulation in purified gonadotropes cell cultures. Endocrinology 1986 118:1148-1158[Abstract/Free Full Text]
  28. Huckle WR, Conn PM. The relationship between gonadotropin-releasing hormone-stimulated luteinizing hormone release and inositol phosphate production: studies with calcium antagonists and protein kinase C activators. Endocrinology 1987 120:160-169[Abstract/Free Full Text]
  29. Sundaresan S, Colin IM, Pestell RG, Jameson JL. Stimulation of mitogen-activated protein kinase by gonadotropin-releasing hormone: evidence for the involvement of protein kinase C. Endocrinology 1996 137:304-311[Abstract]
  30. Parker SL, Kalra SP, Crowley WR. Neuropeptide Y modulates the binding of a gonadotropin-releasing hormone (GnRH) analog to anterior pituitary GnRH receptor sites. Endocrinology 1991 128:2309-2316[Abstract/Free Full Text]




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