Galanin Enhancement of Gonadotropin-Releasing Hormone-Stimulated Luteinizing Hormone Secretion in Female Rats Is Estrogen Dependent
Abstractk(xm, 百拇医药
The hypothalamic peptide GnRH is the primary neuroendocrine signal regulating pituitary LH in females. The neuropeptide galanin is cosecreted with GnRH from hypothalamic neurons, and in vitro studies have demonstrated that galanin can act at the level of the pituitary to directly stimulate LH secretion and also augment GnRH-stimulated LH secretion. Several lines of evidence have suggested that the hypophysiotropic effects of galanin are important for the generation of preovulatory LH surges. To determine whether the pituitary actions of galanin are enhanced by the preovulatory steroidal milieu, LH responses to galanin administration (with or without GnRH) were examined in: 1) ovariectomized (OVX); 2) OVX, estrogen (E)-primed; and 3) OVX, E- and progesterone-treated female rats. Results from the study indicate that galanin enhances GnRH-stimulated LH secretion only in the presence of E (in OVX, E-primed, or E- and progesterone-treated rats). Galanin alone does not directly stimulate LH secretion under any of the steroid conditions examined. In the absence of gonadal steroids (OVX rats), galanin inhibits GnRH-stimulated LH secretion. These findings suggest that the primary pituitary effect of galanin is to modulate GnRH-stimulated LH secretion, and that the potentiating effects of galanin occur only in the presence of E.
Introductionuf]v, 百拇医药
GALANIN IS a 29-amino-acid peptide that was first identified as a gut peptide and subsequently was found to be expressed at high levels throughout the central and peripheral nervous systems of a number of species (1, 2, 3, 4). Galanin immunoreactivity is abundant in the hypothalamus, and there now exists a substantial amount of evidence implicating a role for hypothalamic galanin neurons in the regulation of the reproductive axis. Galanin neurons exert effects on the reproductive axis at two distinct levels. The first of these is at the level of the hypothalamus, where the peptide stimulates the production of the hypothalamic releasing factor GnRH (5, 6, 7). The second is at the level of the pituitary gland, where galanin acts as a hypophysiotropic regulator of pituitary LH secretion (8).uf]v, 百拇医药
The hypothalamic effects of galanin on GnRH secretion were first suggested by the observation that intracerebroventricular injections of galanin increase plasma LH levels in a dose-dependent fashion in steroid-primed, ovariectomized (OVX) rats (5). Subsequent in vitro studies confirmed a role for galanin in the regulation of GnRH release, because the neuropeptide was found to stimulate GnRH release from the hypothalamic fragments of intact male rats (6) and OVX, steroid-primed female rats (7). In females, the effects of galanin on GnRH neurosecretion seem to be important for the generation of preovulatory LH surges, because intracerebroventricular injections of the galanin receptor antagonist galantide result in a complete blockade of LH surges in steroid-primed OVX rats (7).
A role for galanin as a hypophysiotropic regulator of reproductive hormone secretion was first proposed when immunocytochemical studies demonstrated the presence of galanin nerve terminals in the median eminence (9, 10). It was found that, in both sexes, a subset of these neurons coexpresses both galanin and GnRH (11, 12), and portal blood measurements of the two neurohormones in female rats have demonstrated that they are cosecreted into the portal vasculature in a pulsatile manner (8). In vitro examination of the pituitary effects of galanin have demonstrated that its primary effect is to enhance GnRH-stimulated LH secretion, although the peptide can directly stimulate LH secretion on its own when administered at pharmacological doses (8).6q9f, 百拇医药
It has been hypothesized that, like the hypothalamic effects of galanin on GnRH neurosecretion, the pituitary effects of the peptide are important for the generation of preovulatory LH surges in females. This hypothesis is based on the findings that iv administration of the galanin receptor antagonist galantide (7) significantly reduces the amplitude of LH surges in steroid-primed, OVX rats. Furthermore, measurements of galanin mRNA expression in GnRH neurons (13) and galanin content in the median eminence (8) have demonstrated that hypothalamic production of the neuropeptide peaks on the day of proestrus in those galanin neurons that likely serve as hypophysiotropic regulators of LH secretion.
The purpose of the present study was to determine whether the pituitary effects of galanin are enhanced under the steroid conditions in which preovulatory LH surges are generated. A number of studies have demonstrated that pituitary sensitivity to hypothalamic factors involved in the generation of LH surges (e.g. GnRH, neuropeptide Y) is enhanced during the preovulatory period (14, 15, 16). It was hypothesized, in the present study, that if galanin also plays a role in the induction of the LH surges, then gonadotrope sensitivity to the neuropeptide will also be enhanced under the steroid conditions that lead to preovulatory LH surges. To this end, the ability of galanin to directly stimulate LH secretion and/or enhance GnRH-stimulated LH secretion was examined in vivo in OVX; OVX + estrogen (E)-primed; and OVX, E- and progesterone (P)-treated (E+P-treated) rats.l&$4, 百拇医药
Materials and Methodsl&$4, 百拇医药
Animals and surgical protocoll&$4, 百拇医药
All surgical and experimental procedures were conducted in accordance with the policies of University of Wisconsin-Green Bay’s Institutional Review Board. Female Sprague Dawley rats (200–250 g; Charles River Laboratories, Inc., Wilmington, MA) were housed in groups of two to three rats per cage, in a temperature- and humidity-controlled room, with lights on from 0600–2000 h. Animals had access to tap water and standard laboratory rat chow ad libitum.
The pituitary effects of galanin (±GnRH) were examined in three groups of animals: 1) OVX rats; 2) OVX, E-primed rats; and 3) OVX, E+P-treated rats. All surgical procedures (gonadectomies, E capsule and catheter implantations) were performed under Halothane anesthesia (Halocarbon Laboratories, Hackensack, NJ). Animals were bilaterally OVX 7 d before experiments. At 1130 h, 3 d before experiments, a blank or 17ß-estradiol-filled SILASTIC capsule (Dow Corning, Inc. Corp., Midland, MI; 5 mm in length; inside diameter, 0.062 in.; outside diameter, 0.125 in.) was inserted sc.0, http://www.100md.com
On the day before experiments, rats were fitted with indwelling jugular catheters (PE50). The catheter was inserted into the atrium, tunneled sc 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.0, http://www.100md.com
Pulsatile hormone injections and blood sampling
At 1100 h on the morning of experiments, rats received an injection of P (5 mg, sc) or an equivalent volume of sesame oil. From 1200–2100 h, hourly blood samples were withdrawn from the atrial catheter and centrifuged, and the plasma was stored at -20 C. Blood samples were 0.27 ml in vol and were replaced with an equal volume of heparinized saline. At 1330 h, rats received an ip injection of pentobarbital [pento; Sigma-Aldrich Corp., St. Louis, MO; 40 mg/kg body weight (BW)] to block hypothalamic GnRH neurosecretion (17). Control rats received ip injections of an equal volume of 0.9% saline. At 30-min intervals, between 1400 and 1730 h, pento-blocked rats received iv injections of either saline, GnRH alone (Sigma-Aldrich Corp.; 25 ng/pulse), galanin alone (rat galanin; Peninsula Laboratories, Inc., Belmont, CA; 5 or 10 µg/pulse), or galanin (5 or 10 µg/pulse) plus GnRH (25 ng/pulse) (n = 5–6 rats per treatment group). The two doses of galanin were chosen to produce peak peripheral blood concentrations that were 5-fold or 10-fold greater than concentrations observed in portal vessel blood at the peak of a galanin pulse (8). The dose of GnRH used in the study was determined, in preliminary experiments, to produce an LH surge in pento-blocked, OVX, E+P-treated rats that was comparable in magnitude with the LH surge observed in control animals. Both GnRH and galanin were administered in a pulsatile manner to mimic the endogenous secretory profiles of the peptides (8). 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.
RIAs.ha[, 百拇医药
LH levels in plasma samples were determined by 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 assay was 0.28 ng/ml..ha[, 百拇医药
Statistical analysis of data.ha[, 百拇医药
For each animal, LH levels in samples collected from 1500–1800 h were averaged. Group means of LH levels were generated, and Student’s t test was used to assess differences between saline- vs. pento-treated control groups (animals that received saline pulses alone between 1400 and 1730 h) within a particular steroid treatment group (OVX; OVX+E; or OVX, E+P-treated animals). One-way ANOVA was used to assess differences in mean LH levels in pento-blocked animals in response to hormone treatments (saline, galanin alone, or galanin + GnRH) within a particular steroid treatment group (OVX; OVX + E; or OVX, E+P-treated animals). Post hoc comparisons between mean values for each hormone treatment were made using Tukey’s least-significant-difference test. Results were considered significant at P < 0.05.
Results(/wow7, 百拇医药
Effects of pento on LH release in OVX rats(/wow7, 百拇医药
OVX rats treated with blank capsules, an sc injection of oil at 1100 h, an ip injection of vehicle at 1330 h, and iv pulses of saline from 1400–1730 h exhibited a mean plasma LH level of 3.44 ± 0.60 ng/ml (mean ± SE) from 1500–1800 h (n = 5 for this and subsequent groups, unless noted otherwise). Administration of pento at 1330 h to saline-treated OVX rats resulted in a significant decline in plasma LH levels (1.96 ± 0.31 ng/ml; P < 0.05; see Fig. 1A). This decline in LH levels was a result of the barbiturate’s inhibitory influence on hypothalamic GnRH neurosecretion (17).(/wow7, 百拇医药
fig.ommitteedfig.ommitteed(/wow7, 百拇医药
Figure 1. A, Effects of ip administration of pento (40 mg/kg BW) on plasma LH levels in OVX rats. B, Effects of iv galanin administration (5 or 10 µg/pulse) on plasma LH levels in OVX, pento-blocked rats. C, Effects of coadministration of galanin (5 or 10 µg/pulse) and GnRH (25 ng/pulse) on plasma LH levels in OVX, pento-blocked rats. In all graphs, arrows denote times of ip administration of pento or saline, and solid bars denote time periods during which pulsatile infusions of hormones (or saline) took place. Note the difference in scale of the y-axis in C.
Effects of galanin alone on LH release in pento-blocked, OVX rats(+#6p, http://www.100md.com
Pento-blocked, OVX rats that received no further steroid treatment exhibited a mean plasma LH level of 1.76 ± 0.15 ng/ml between 1500 and 1800 h, in response to administration of 5 µg/pulse galanin from 1400–1730 h. This mean plasma LH level did not differ significantly from the level observed in pento-blocked, saline-treated, OVX animals. Likewise, LH levels in pento-blocked, OVX rats treated with galanin alone at a dose of 10 µg/pulse (2.37 ± 0.38 ng/ml) did not differ significantly from levels observed in saline-treated control animals (see Fig. 1B).(+#6p, http://www.100md.com
Effects of galanin on GnRH-stimulated LH release in pento-blocked, OVX rats(+#6p, http://www.100md.com
Pulsatile administration of GnRH alone (25 ng/pulse) to pento-blocked, OVX rats caused a significant elevation in mean plasma LH levels from 1500–1800 h (7.18 ± 0.75 ng/ml; n = 6) when compared with levels observed in saline-treated controls (P < 0.001). Coadministration of galanin at either dose significantly inhibited the ability of 25 ng/pulse GnRH to stimulate LH secretion in pento-blocked, OVX rats. OVX rats treated concomitantly with 5 µg/pulse galanin and 25 ng/pulse GnRH exhibited a mean plasma LH level of 4.28 ± 0.33 ng/ml (n = 6) from 1500–1800 h, while OVX rats treated concomitantly with 10 µg/pulse galanin and 25 ng/pulse GnRH exhibited a mean plasma LH level of 2.37 ± 0.24 ng/ml from 1500–1800 h (n = 6). Both of these mean LH levels were significantly lower than levels observed in pento-blocked, OVX rats treated with 25 ng/pulse GnRH alone (P < 0.005; see Fig. 1C).
Effects of pento on LH secretion in OVX, E-primed rats$2!^}, http://www.100md.com
Figure 2A illustrates that the regimen of E-priming used in the present study induced a modest LH surge in OVX rats. OVX, E-primed rats treated with an sc injection of oil at 1100 h, an ip injection of vehicle at 1330 h, and iv pulses of saline from 1400–1730 h exhibited a mean plasma LH level of 5.07 ± 1.23 ng/ml between 1500 and 1800 h (n = 6). Treatment with an injection of pento at 1330 h (rather than vehicle) effectively blocked the GnRH (and thus LH) surge in these animals; mean plasma LH levels in pento-blocked, OVX, E-primed rats treated with pulses of saline alone declined to 1.01 ± 0.27 ng/ml between 1500 and 1800 h (P < 0.05).$2!^}, http://www.100md.com
fig.ommitteedfig.ommitteed$2!^}, http://www.100md.com
Figure 2. A, Effects of ip administration of pento (40 mg/kg BW) on plasma LH levels in OVX, E-primed rats. B, Effects of iv galanin administration (5 or 10 µg/pulse) on plasma LH levels in OVX, E-primed, pento-blocked rats. C, Effects of coadministration of galanin (5 or 10 µg/pulse) and GnRH (25 ng/pulse) on plasma LH levels in OVX, E-primed, pento-blocked rats. Note the difference in scale of the y-axis in C.
Effects of galanin alone on LH release in pento-blocked, OVX, E-primed ratsyj/ge-, 百拇医药
Neither dose of galanin used in the present study was capable of stimulating LH secretion on its own in OVX, E-primed rats. Administration of galanin alone at a dose of 5 µg/pulse resulted in a mean LH level of 1.46 ± 0.15 ng/ml from 1500–1800 h (n = 6), whereas administration of galanin alone at a dose of 10 µg/pulse resulted in mean a plasma LH level of 0.93 ± 0.12 ng/ml between 1500 and 1800 h. Neither of these mean LH levels differed significantly from those observed in pento-blocked, OVX, E-primed rats treated with saline only from 1400–1730 h (see Fig. 2B).yj/ge-, 百拇医药
Effects of galanin on GnRH-stimulated LH secretion in pento-blocked, OVX, E-primed ratsyj/ge-, 百拇医药
Pulsatile administration of GnRH at a dose of 25 ng/pulse resulted in a mean LH level of 27.93 ± 1.58 ng/ml between 1500 and 1800 h in pento-blocked, OVX, E-primed rats (n = 6; see Fig. 2C). This mean LH level was significantly greater than levels observed in pento-blocked, OVX, E-primed rats treated with saline pulses from 1500–1800 h (P < 0.001). Coadministration of galanin at either 5 or 10 µg/pulse resulted in a significant potentiation of GnRH-stimulated LH secretion in OVX, E-primed animals. Animals treated with GnRH + 5 µg galanin/pulse exhibited a mean plasma LH level of 36.32 ± 5.07 ng/ml between 1500 and 1800 h, a mean level that was 1.3-fold greater than levels observed in response to treatment with the same dose of GnRH alone (P < 0.05). Likewise, coadministration of GnRH +10 µg galanin/pulse resulted in a mean plasma LH level of 39.11 ± 3.34 ng/ml between 1500 and 1800 h, a mean level that was 1.4-fold greater than levels observed in animals treated with the same dose of GnRH alone (P < 0.005; see Fig. 2C).
Effects of pento on LH secretion in OVX, E+P-treated ratsy3y+, 百拇医药
OVX, E-primed rats treated with P at 1100 h, an ip injection of saline at 1330 h, and pulses of saline from 1400–1730 h exhibited an LH surge on the afternoon of experiments. The mean LH level in OVX, E+P-treated control animals was 12.56 ± 3.05 ng/ml between 1500 and 1800 h (n = 6; see Fig. 3A). Administration of an ip injection of pento at 1330 h effectively blocked the steroid-induced LH surge in OVX, E+P-treated animals; mean plasma LH levels in these animals were 0.82 ± 0.22 ng/ml from 1500–1800 h, a mean level that was significantly lower than that observed in OVX, E+P-treated controls (P < 0.05).y3y+, 百拇医药
fig.ommitteedfig.ommitteedy3y+, 百拇医药
Figure 3. A, Effects of ip administration of pento (40 mg/kg BW) on plasma LH levels in OVX, E+P-treated rats. B, Effects of iv galanin administration (5 or 10 µg/pulse) on plasma LH levels in OVX, E+P-treated, pento-blocked rats. C, Effects of coadministration of galanin (5 or 10 µg/pulse) and GnRH (25 ng/pulse) on plasma LH levels in OVX, E+P-treated, pento-blocked rats. Note the difference in scale of the y-axis in C.
Effects of galanin alone on LH release in pento-blocked, OVX, E+P-treated ratso/i83i@, 百拇医药
Pulsatile administration of galanin alone at either dose used in the present study had no effect on LH secretion in OVX, E+P-treated rats. The mean LH level in OVX, E+P-treated rats treated with a 5 µg/pulse of galanin from 1400–1730 h was 1.47 ± 0.25 ng/ml; this mean LH level did not differ significantly from that observed in OVX, E+P-treated, pento-blocked rats that received pulses of saline alone from 1400–1730 h. Likewise, treatment of OVX, E+P-treated rats with 10 µg/pulse of galanin resulted in a mean LH level of 0.77 ± 0.12 ng/ml, a mean LH level that did not differ significantly from levels observed in OVX, E+P-treated, pento-blocked control animals (see Fig. 3B).o/i83i@, 百拇医药
Effects of galanin on GnRH-stimulated LH secretion in pento-blocked, OVX, E+P-treated ratso/i83i@, 百拇医药
Pulsatile administration of GnRH at a dose of 25 ng/pulse to pento-blocked, OVX, E+P-treated rats caused a significant elevation in mean plasma LH levels when compared with LH levels in pento-blocked, saline-treated control rats within the same steroid treatment group (29.07 ± 3.82 ng/ml; n = 6; P < 0.001; see Fig. 3C. Coadministration of either dose of galanin with 25 ng/pulse of GnRH caused a significant potentiation of GnRH-stimulated LH secretion in OVX, E+P-treated rats. Combined infusions of galanin at a dose of 5 µg/pulse and GnRH at a dose of 25 ng/pulse resulted in a mean plasma LH level of 40.58 ± 7.08 ng/ml between 1500 and 1800 h, a level that was 1.4-fold greater than levels observed in OVX, E+P-treated animals that received the same dose of GnRH alone (P < 0.05). Combined infusions of galanin at a dose of 10 µg/pulse along with GnRH at a dose of 25 ng/pulse resulted in a mean plasma LH level of 40.75 ± 5.49 ng/ml between 1500 and 1800 h, a mean level that was also 1.4-fold greater than that observed in animals treated with the same dose of GnRH alone (P < 0.05; see Fig. 3C).
Discussiong, 百拇医药
A variety of experimental evidence now supports the notion that hypothalamic galanin neurons participate in the induction of preovulatory LH surges in female rats. During the female rat estrous cycle, galanin mRNA expression in GnRH neurons (13) and galanin content in the basal hypothalamus (8) are highest on the day of proestrus in female rats, indicating an activation of hypothalamic galanin systems within the context of the preovulatory endocrine milieu. Elevated levels of estrogen on the day of proestrus are thought to be responsible for the up-regulation of galanin production at this time, given that estrogen has been shown to induce galanin mRNA and protein expression in hypothalamic neurons under a variety of experimental circumstances (18, 19, 20). Increased hypothalamic galanin production likely contributes to the generation of preovulatory LH surges at both the hypothalamic and pituitary levels on the day of proestrus, by participating in the generation of the GnRH surge and enhancing GnRH-stimulated LH secretion, respectively. These hypotheses are based on the findings that both central and peripheral blockade of galanin’s actions attenuates steroid-induced LH surges in female rats (7).
The results of the present study provide evidence that the pituitary actions of galanin are enhanced under the endocrine conditions in which preovulatory LH surges are generated. Galanin was found to significantly enhance GnRH-stimulated LH secretion within the context of the preovulatory steroidal milieu; coadministration of galanin along with GnRH produced LH surges in steroid-primed animals that were 30–40% greater than LH surges observed in animals treated with the same dose of GnRH alone. This effect of galanin was found to be strictly modulatory in nature, in that pulsatile administration of galanin alone had no effect on LH secretion in steroid-primed animals. Comparisons of the peptide’s neuromodulatory effects across steroid treatment groups in the present study indicate that estrogen is responsible for up-regulating pituitary sensitivity to galanin, because galanin enhanced GnRH-stimulated LH secretion equally in OVX, E-primed and OVX, E+P-treated rats but had no potentiating effect in OVX, unprimed animals. These results suggest that, as galanin production within hypothalamic neurons increases on proestrus, pituitary sensitivity to the neuropeptide is also up-regulated—just as it is for other neuropeptides involved in the generation of LH surges (GnRH, neuropeptide Y; 14–16)—thereby ensuring a robust LH surge and ultimately ovulation.
Interestingly, our laboratory recently demonstrated that galanin also acts to potentiate GnRH-stimulated LH secretion in male rats; and, like the effects observed in female rats in the present study, the pituitary effects of galanin in males are steroid-dependent (21). It is likely that the GALR2 receptor subtype mediates the modulatory effects of galanin on GnRH-stimulated LH secretion in males, given that combined in situ hybridization immunohistochemistry studies have demonstrated the presence of GALR2 mRNA in pituitary gonadotropes (22). Whether the GALR2 receptor is expressed in gonadotropes of the female pituitary gland and mediates galanin’s effects on GnRH-stimulated LH secretion in females remains to be determined; however, it is interesting to note that the galanin receptor antagonist galantide, which attenuates steroid-induced LH surges when administered peripherally to OVX rats, is known to bind to the rat GALR2 receptor (23).h?34, http://www.100md.com
The cellular mechanism(s) whereby galanin enhances pituitary sensitivity to GnRH in steroid-primed animals is also unknown. Galanin may induce changes in GnRH receptor number and/or affinity or may augment the intracellular signal transduction mechanisms coupled to the GnRH receptor. There is evidence to suggest that galanin can modulate GnRH binding to anterior pituitary membranes obtained from chronically OVX rats (24). However, the effects observed were modest and occurred in response to treatment with a pharmacological dose of the peptide. Further studies will be necessary to determine the extent to which galanin can enhance GnRH binding to gonadotropes exposed to proestrous levels of estrogen. Because the galanin receptor subtype expressed on gonadotropes in female rats remains largely uncharacterized at this point in time, discussion of whether galanin receptor activation augments the signal transduction pathways mediating GnRH-stimulated LH secretion remains purely speculative. However, it is interesting to note that the GALR2 receptor, which is expressed in gonadotropes in the male (22), is capable of stimulating inositol phospholipid turnover, mobilizing intracellular calcium (25, 26), and stimulating MAPK activity (25), and all of these signal transduction mechanisms are known to be activated by the GnRH receptor in pituitary gonadotropes (27, 28, 29). It is noteworthy that the potentiating effects of galanin on GnRH-stimulated LH secretion in both OVX, E-primed and OVX, E+P-treated animals were not evident until 1600 h on the afternoon of experiments in the present study. Given that pulsatile hormone administration was initiated at 1400 h, this observation would suggest that there is a 2-h lag in time between exposure to galanin and the onset of the peptide’s potentiating effect on GnRH-stimulated LH secretion.
The ability of galanin to potentiate GnRH-stimulated LH secretion in OVX, E-primed and OVX, E+P-treated animals was not found to be dose-dependent in the present study. In these two steroid treatment groups, mean LH levels did not differ significantly between animals treated with GnRH + 5 µg/pulse galanin and GnRH +10 µg/pulse galanin. A likely explanation for this lack of dose-dependence may be that, on coadministration of the lower dose of galanin along with GnRH, gonadotropes are responding maximally to stimulation by the neuropeptides, and thus, administration of a higher dose of galanin along with GnRH produces no further increase in LH secretion. It is noteworthy, in the present study, that P treatment resulted in no further enhancement of galanin’s effects on GnRH-stimulated LH secretion in estrogen-primed, OVX rats. This result contradicts the findings of Sanchez-Criado et al. (30), who observed that the antiprogestin RU486 blunts the stimulatory effects of galanin on GnRH-stimulated LH secretion from proestrous pituitaries in vitro. Although P treatment did not further enhance galanin’s effects on GnRH-stimulated LH secretion in estrogen-primed, OVX rats in the present study, it did shift the time course of the GnRH-induced LH surge, in that the peak of the surge occurred at 1500 h in animals treated with GnRH alone vs. 1600 h in animals treated with GnRH + 5 or 10 µg/pulse of galanin. It is anticipated that further experiments regarding the intracellular mechanism(s) whereby galanin enhances GnRH-stimulated LH secretion, and the impact of gonadal steroids on galanin receptor-induced signal transduction in gonadotropes, will reveal the significance of this phenomenon.
Interestingly, not only were the potentiating effects of galanin absent in OVX rats in the present study, but the peptide was actually found to inhibit GnRH-stimulated LH secretion in the complete absence of gonadal steroids. A similar inhibitory effect of galanin on GnRH-stimulated LH secretion has been observed in castrated male rats (21) and in anterior pituitaries harvested from proestrus female rats and cultured in the absence of estrogen (31). Taken together, results from these studies suggest that removal of gonadal steroid feedback unmasks an inhibitory effect of galanin on GnRH-stimulated LH secretion. Further in vivo studies will be necessary to determine the physiological significance of this inhibitory phenomenon, and to examine whether it occurs only in the complete absence of estrogen (i.e. in gonadectomized animals), or whether it exists in females at times during the estrous cycle when circulating levels of estrogen are low (i.e. in the presence of negative feedback levels of estrogen). Intriguingly, Cardenas et al. (32) observed, in their in vitro studies, that galanin is capable of potentiating GnRH-stimulated LH secretion from hemipituitary fragments obtained from diestrus 1 rats. This finding would suggest that the potentiating effects of galanin are maintained in the presence of low levels of estrogen, and that the inhibitory effects of the peptide, if present under physiological circumstances, are limited to conditions of hypogonadism.
In addition to hypothalamic neurons serving as a source of galanin, immunocytochemical studies have demonstrated that the anterior pituitary itself synthesizes a significant quantity of galanin (33). Immunoreactive galanin in the pituitary gland is mainly localized to lactotropes (34), and studies in female rats have demonstrated that estrogen is a potent stimulator of both galanin mRNA expression and protein synthesis in lactotropes (33, 35). Given that lactotropes exist within close proximity to gonadotropes within the dorsocephalic region of the anterior pituitary gland (36, 37), it is likely that lactotropes (in addition to hypothalamic galanin neurons) serve as a significant source of galanin at times of estrous cycle when estrogen levels are high (i.e. during the preovulatory period). Pituitary galanin may then act in a paracrine manner to enhance pituitary sensitivity to GnRH at the time of the preovulatory LH surge.h'[m', http://www.100md.com
It is now widely recognized that at the time of the preovulatory LH surge, the anterior pituitary gland is 50 times more sensitive to GnRH stimulation than it is during the rest of the estrous cycle (14). This acute increase in pituitary responsiveness to GnRH stimulation has been attributed to a number of factors, including GnRH self-priming (38), the direct effects of adrenal or ovarian P on the pituitary gland (39, 40), and the priming actions of neuropeptide Y (41). The results from the present study indicate that the neuropeptide galanin should also be added to this growing list of factors that act to ensure the efficacy of the GnRH—and hence, the LH—surge, and ultimately ovulation.
In conclusion, we have demonstrated that galanin significantly enhances GnRH-stimulated LH secretion in the presence of high levels of estrogen (in OVX, E-primed and OVX, E+P-treated animals). Galanin does not potentiate GnRH-stimulated LH secretion in OVX, unprimed animals; and, in fact, the neuropeptide inhibits GnRH-stimulated LH secretion in the absence of gonadal steroids. Administration of galanin alone (without GnRH) has no effect on LH secretion under any of the steroid treatment conditions examined in the present study. These results strongly implicate a role for galanin in the up-regulation of pituitary sensitivity to GnRH at the time of the preovulatory LH surge in female rats. Further studies are necessary to delineate the cellular mechanism(s) whereby galanin exerts its modulatory effects in pituitary gonadotropes.eceived August 15, 2002.;7, 百拇医药
Accepted for publication October 15, 2002.;7, 百拇医药
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Merchenthaler I 1991 The hypophysiotropic galanin system of the rat brain. Neuroscience 44:643–668w, 百拇医药
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Marks DL, Smith MS, Clifton DK, Steiner RA 1993 Regulation of galanin gene expression in gonadotropin-releasing hormone neurons during the estrous cycle of the rat. Endocrinology 132:1836–1843
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Bauer-Dantoin AC, McDonald JK, Levine JE 1991 Neuropeptide Y potentiates luteinizing hormone (LH)-releasing hormone-stimulated LH surges in pentobarbital-blocked, proestrous rats. Endocrinology 129:402–4085[k0r8l, 百拇医药
Bauer-Dantoin AC, McDonald JK, Levine JE 1993 Neuropeptide Y potentiates LHRH-induced LH secretion only under conditions leading to preovulatory LH surges. Endocrinology 131:2946–29525[k0r8l, 百拇医药
Ching M 1982 Correlative surges of LHRH, LH and FSH in pituitary stalk plasma and systemic plasma of rat during proestrus. Neuroendocrinology 34:279–2855[k0r8l, 百拇医药
Ceresini G, Merchenthaler A, Negro-Vilar A, Merchenthaler I 1994 Aging impairs galanin expression in luteinizing hormone-releasing hormone neurons: effect of ovariectomy and/or estradiol treatment. Endocrinology 134:324–4405[k0r8l, 百拇医药
Liposits Z, Reid JJ, Negro-Vilar A, Merchenthaler I 1995 Sexual dimorphism in copackaging of luteinizing hormone-releasing hormone and galanin into neurosecretory vesicles of hypophysiotrophic neurons: estrogen dependency. Endocrinology 136:1987–1992
Shen ES, Meade EH, Perez MC, Deecher DC, Negro-Vilar A, Lopez FJ 1998 Expression of functional estrogen receptors and galanin messenger ribonucleic acid in immortalized luteinizing hormone-releasing hormone neurons: estrogenic control of galanin gene expression. Endocrinology 139:939–948{dmoy, http://www.100md.com
Scheffen JR, Splett CL, Desotelle JA, Bauer-Dantoin AC, Testosterone-dependent effects of galanin on pituitary luteinizing hormone secretion in male rats. Biol Reprod, in press{dmoy, http://www.100md.com
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Sanchez-Criado JE, Bellido C, Aguilar R, Garrido-Gracia JC 2001 Antiprogestin RU486 blunts the stimulatory effect of galanin on LH secretion from rat pituitaries in vitro in an estrous cycle stage-dependent manner. Neurosci Lett 305:73–7567, http://www.100md.com
Todd JF, Small CJ, Akinsanya KO, Stanley SA, Smith DM, Bloom SR 1998 Galanin is a paracrine inhibitor of gonadotroph function in the female rat. Endocrinology 139:4222–422967, http://www.100md.com
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Kaplan LM, Gabriel SM, Koenig JI, Sunday ME, Spindel ER, Martin JB, Chin WW 1988 Galanin is an estrogen-inducible, secretory product of the rat anterior pituitary. Proc Natl Acad Sci USA 85:7408–741267, http://www.100md.com
Steel JH, Gon G, O’Halloran DJ, Jones PM, Yanaihara N, Ishikawa H, Bloom SR, Polak JM 1989 Galanin and vasoactive intestinal polypeptide are colocalised with classical pituitary hormones and show plasticity of expression. Histochemistry 93:183–189
Vrontakis ME, Schroedter IC, Crosby H, Friesen HG 1992 Expression and secretion of galanin during pregnancy in the rat. Endocrinology 130:458–4642au0]i6, http://www.100md.com
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The hypothalamic peptide GnRH is the primary neuroendocrine signal regulating pituitary LH in females. The neuropeptide galanin is cosecreted with GnRH from hypothalamic neurons, and in vitro studies have demonstrated that galanin can act at the level of the pituitary to directly stimulate LH secretion and also augment GnRH-stimulated LH secretion. Several lines of evidence have suggested that the hypophysiotropic effects of galanin are important for the generation of preovulatory LH surges. To determine whether the pituitary actions of galanin are enhanced by the preovulatory steroidal milieu, LH responses to galanin administration (with or without GnRH) were examined in: 1) ovariectomized (OVX); 2) OVX, estrogen (E)-primed; and 3) OVX, E- and progesterone-treated female rats. Results from the study indicate that galanin enhances GnRH-stimulated LH secretion only in the presence of E (in OVX, E-primed, or E- and progesterone-treated rats). Galanin alone does not directly stimulate LH secretion under any of the steroid conditions examined. In the absence of gonadal steroids (OVX rats), galanin inhibits GnRH-stimulated LH secretion. These findings suggest that the primary pituitary effect of galanin is to modulate GnRH-stimulated LH secretion, and that the potentiating effects of galanin occur only in the presence of E.
Introductionuf]v, 百拇医药
GALANIN IS a 29-amino-acid peptide that was first identified as a gut peptide and subsequently was found to be expressed at high levels throughout the central and peripheral nervous systems of a number of species (1, 2, 3, 4). Galanin immunoreactivity is abundant in the hypothalamus, and there now exists a substantial amount of evidence implicating a role for hypothalamic galanin neurons in the regulation of the reproductive axis. Galanin neurons exert effects on the reproductive axis at two distinct levels. The first of these is at the level of the hypothalamus, where the peptide stimulates the production of the hypothalamic releasing factor GnRH (5, 6, 7). The second is at the level of the pituitary gland, where galanin acts as a hypophysiotropic regulator of pituitary LH secretion (8).uf]v, 百拇医药
The hypothalamic effects of galanin on GnRH secretion were first suggested by the observation that intracerebroventricular injections of galanin increase plasma LH levels in a dose-dependent fashion in steroid-primed, ovariectomized (OVX) rats (5). Subsequent in vitro studies confirmed a role for galanin in the regulation of GnRH release, because the neuropeptide was found to stimulate GnRH release from the hypothalamic fragments of intact male rats (6) and OVX, steroid-primed female rats (7). In females, the effects of galanin on GnRH neurosecretion seem to be important for the generation of preovulatory LH surges, because intracerebroventricular injections of the galanin receptor antagonist galantide result in a complete blockade of LH surges in steroid-primed OVX rats (7).
A role for galanin as a hypophysiotropic regulator of reproductive hormone secretion was first proposed when immunocytochemical studies demonstrated the presence of galanin nerve terminals in the median eminence (9, 10). It was found that, in both sexes, a subset of these neurons coexpresses both galanin and GnRH (11, 12), and portal blood measurements of the two neurohormones in female rats have demonstrated that they are cosecreted into the portal vasculature in a pulsatile manner (8). In vitro examination of the pituitary effects of galanin have demonstrated that its primary effect is to enhance GnRH-stimulated LH secretion, although the peptide can directly stimulate LH secretion on its own when administered at pharmacological doses (8).6q9f, 百拇医药
It has been hypothesized that, like the hypothalamic effects of galanin on GnRH neurosecretion, the pituitary effects of the peptide are important for the generation of preovulatory LH surges in females. This hypothesis is based on the findings that iv administration of the galanin receptor antagonist galantide (7) significantly reduces the amplitude of LH surges in steroid-primed, OVX rats. Furthermore, measurements of galanin mRNA expression in GnRH neurons (13) and galanin content in the median eminence (8) have demonstrated that hypothalamic production of the neuropeptide peaks on the day of proestrus in those galanin neurons that likely serve as hypophysiotropic regulators of LH secretion.
The purpose of the present study was to determine whether the pituitary effects of galanin are enhanced under the steroid conditions in which preovulatory LH surges are generated. A number of studies have demonstrated that pituitary sensitivity to hypothalamic factors involved in the generation of LH surges (e.g. GnRH, neuropeptide Y) is enhanced during the preovulatory period (14, 15, 16). It was hypothesized, in the present study, that if galanin also plays a role in the induction of the LH surges, then gonadotrope sensitivity to the neuropeptide will also be enhanced under the steroid conditions that lead to preovulatory LH surges. To this end, the ability of galanin to directly stimulate LH secretion and/or enhance GnRH-stimulated LH secretion was examined in vivo in OVX; OVX + estrogen (E)-primed; and OVX, E- and progesterone (P)-treated (E+P-treated) rats.l&$4, 百拇医药
Materials and Methodsl&$4, 百拇医药
Animals and surgical protocoll&$4, 百拇医药
All surgical and experimental procedures were conducted in accordance with the policies of University of Wisconsin-Green Bay’s Institutional Review Board. Female Sprague Dawley rats (200–250 g; Charles River Laboratories, Inc., Wilmington, MA) were housed in groups of two to three rats per cage, in a temperature- and humidity-controlled room, with lights on from 0600–2000 h. Animals had access to tap water and standard laboratory rat chow ad libitum.
The pituitary effects of galanin (±GnRH) were examined in three groups of animals: 1) OVX rats; 2) OVX, E-primed rats; and 3) OVX, E+P-treated rats. All surgical procedures (gonadectomies, E capsule and catheter implantations) were performed under Halothane anesthesia (Halocarbon Laboratories, Hackensack, NJ). Animals were bilaterally OVX 7 d before experiments. At 1130 h, 3 d before experiments, a blank or 17ß-estradiol-filled SILASTIC capsule (Dow Corning, Inc. Corp., Midland, MI; 5 mm in length; inside diameter, 0.062 in.; outside diameter, 0.125 in.) was inserted sc.0, http://www.100md.com
On the day before experiments, rats were fitted with indwelling jugular catheters (PE50). The catheter was inserted into the atrium, tunneled sc 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.0, http://www.100md.com
Pulsatile hormone injections and blood sampling
At 1100 h on the morning of experiments, rats received an injection of P (5 mg, sc) or an equivalent volume of sesame oil. From 1200–2100 h, hourly blood samples were withdrawn from the atrial catheter and centrifuged, and the plasma was stored at -20 C. Blood samples were 0.27 ml in vol and were replaced with an equal volume of heparinized saline. At 1330 h, rats received an ip injection of pentobarbital [pento; Sigma-Aldrich Corp., St. Louis, MO; 40 mg/kg body weight (BW)] to block hypothalamic GnRH neurosecretion (17). Control rats received ip injections of an equal volume of 0.9% saline. At 30-min intervals, between 1400 and 1730 h, pento-blocked rats received iv injections of either saline, GnRH alone (Sigma-Aldrich Corp.; 25 ng/pulse), galanin alone (rat galanin; Peninsula Laboratories, Inc., Belmont, CA; 5 or 10 µg/pulse), or galanin (5 or 10 µg/pulse) plus GnRH (25 ng/pulse) (n = 5–6 rats per treatment group). The two doses of galanin were chosen to produce peak peripheral blood concentrations that were 5-fold or 10-fold greater than concentrations observed in portal vessel blood at the peak of a galanin pulse (8). The dose of GnRH used in the study was determined, in preliminary experiments, to produce an LH surge in pento-blocked, OVX, E+P-treated rats that was comparable in magnitude with the LH surge observed in control animals. Both GnRH and galanin were administered in a pulsatile manner to mimic the endogenous secretory profiles of the peptides (8). 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.
RIAs.ha[, 百拇医药
LH levels in plasma samples were determined by 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 assay was 0.28 ng/ml..ha[, 百拇医药
Statistical analysis of data.ha[, 百拇医药
For each animal, LH levels in samples collected from 1500–1800 h were averaged. Group means of LH levels were generated, and Student’s t test was used to assess differences between saline- vs. pento-treated control groups (animals that received saline pulses alone between 1400 and 1730 h) within a particular steroid treatment group (OVX; OVX+E; or OVX, E+P-treated animals). One-way ANOVA was used to assess differences in mean LH levels in pento-blocked animals in response to hormone treatments (saline, galanin alone, or galanin + GnRH) within a particular steroid treatment group (OVX; OVX + E; or OVX, E+P-treated animals). Post hoc comparisons between mean values for each hormone treatment were made using Tukey’s least-significant-difference test. Results were considered significant at P < 0.05.
Results(/wow7, 百拇医药
Effects of pento on LH release in OVX rats(/wow7, 百拇医药
OVX rats treated with blank capsules, an sc injection of oil at 1100 h, an ip injection of vehicle at 1330 h, and iv pulses of saline from 1400–1730 h exhibited a mean plasma LH level of 3.44 ± 0.60 ng/ml (mean ± SE) from 1500–1800 h (n = 5 for this and subsequent groups, unless noted otherwise). Administration of pento at 1330 h to saline-treated OVX rats resulted in a significant decline in plasma LH levels (1.96 ± 0.31 ng/ml; P < 0.05; see Fig. 1A). This decline in LH levels was a result of the barbiturate’s inhibitory influence on hypothalamic GnRH neurosecretion (17).(/wow7, 百拇医药
fig.ommitteedfig.ommitteed(/wow7, 百拇医药
Figure 1. A, Effects of ip administration of pento (40 mg/kg BW) on plasma LH levels in OVX rats. B, Effects of iv galanin administration (5 or 10 µg/pulse) on plasma LH levels in OVX, pento-blocked rats. C, Effects of coadministration of galanin (5 or 10 µg/pulse) and GnRH (25 ng/pulse) on plasma LH levels in OVX, pento-blocked rats. In all graphs, arrows denote times of ip administration of pento or saline, and solid bars denote time periods during which pulsatile infusions of hormones (or saline) took place. Note the difference in scale of the y-axis in C.
Effects of galanin alone on LH release in pento-blocked, OVX rats(+#6p, http://www.100md.com
Pento-blocked, OVX rats that received no further steroid treatment exhibited a mean plasma LH level of 1.76 ± 0.15 ng/ml between 1500 and 1800 h, in response to administration of 5 µg/pulse galanin from 1400–1730 h. This mean plasma LH level did not differ significantly from the level observed in pento-blocked, saline-treated, OVX animals. Likewise, LH levels in pento-blocked, OVX rats treated with galanin alone at a dose of 10 µg/pulse (2.37 ± 0.38 ng/ml) did not differ significantly from levels observed in saline-treated control animals (see Fig. 1B).(+#6p, http://www.100md.com
Effects of galanin on GnRH-stimulated LH release in pento-blocked, OVX rats(+#6p, http://www.100md.com
Pulsatile administration of GnRH alone (25 ng/pulse) to pento-blocked, OVX rats caused a significant elevation in mean plasma LH levels from 1500–1800 h (7.18 ± 0.75 ng/ml; n = 6) when compared with levels observed in saline-treated controls (P < 0.001). Coadministration of galanin at either dose significantly inhibited the ability of 25 ng/pulse GnRH to stimulate LH secretion in pento-blocked, OVX rats. OVX rats treated concomitantly with 5 µg/pulse galanin and 25 ng/pulse GnRH exhibited a mean plasma LH level of 4.28 ± 0.33 ng/ml (n = 6) from 1500–1800 h, while OVX rats treated concomitantly with 10 µg/pulse galanin and 25 ng/pulse GnRH exhibited a mean plasma LH level of 2.37 ± 0.24 ng/ml from 1500–1800 h (n = 6). Both of these mean LH levels were significantly lower than levels observed in pento-blocked, OVX rats treated with 25 ng/pulse GnRH alone (P < 0.005; see Fig. 1C).
Effects of pento on LH secretion in OVX, E-primed rats$2!^}, http://www.100md.com
Figure 2A illustrates that the regimen of E-priming used in the present study induced a modest LH surge in OVX rats. OVX, E-primed rats treated with an sc injection of oil at 1100 h, an ip injection of vehicle at 1330 h, and iv pulses of saline from 1400–1730 h exhibited a mean plasma LH level of 5.07 ± 1.23 ng/ml between 1500 and 1800 h (n = 6). Treatment with an injection of pento at 1330 h (rather than vehicle) effectively blocked the GnRH (and thus LH) surge in these animals; mean plasma LH levels in pento-blocked, OVX, E-primed rats treated with pulses of saline alone declined to 1.01 ± 0.27 ng/ml between 1500 and 1800 h (P < 0.05).$2!^}, http://www.100md.com
fig.ommitteedfig.ommitteed$2!^}, http://www.100md.com
Figure 2. A, Effects of ip administration of pento (40 mg/kg BW) on plasma LH levels in OVX, E-primed rats. B, Effects of iv galanin administration (5 or 10 µg/pulse) on plasma LH levels in OVX, E-primed, pento-blocked rats. C, Effects of coadministration of galanin (5 or 10 µg/pulse) and GnRH (25 ng/pulse) on plasma LH levels in OVX, E-primed, pento-blocked rats. Note the difference in scale of the y-axis in C.
Effects of galanin alone on LH release in pento-blocked, OVX, E-primed ratsyj/ge-, 百拇医药
Neither dose of galanin used in the present study was capable of stimulating LH secretion on its own in OVX, E-primed rats. Administration of galanin alone at a dose of 5 µg/pulse resulted in a mean LH level of 1.46 ± 0.15 ng/ml from 1500–1800 h (n = 6), whereas administration of galanin alone at a dose of 10 µg/pulse resulted in mean a plasma LH level of 0.93 ± 0.12 ng/ml between 1500 and 1800 h. Neither of these mean LH levels differed significantly from those observed in pento-blocked, OVX, E-primed rats treated with saline only from 1400–1730 h (see Fig. 2B).yj/ge-, 百拇医药
Effects of galanin on GnRH-stimulated LH secretion in pento-blocked, OVX, E-primed ratsyj/ge-, 百拇医药
Pulsatile administration of GnRH at a dose of 25 ng/pulse resulted in a mean LH level of 27.93 ± 1.58 ng/ml between 1500 and 1800 h in pento-blocked, OVX, E-primed rats (n = 6; see Fig. 2C). This mean LH level was significantly greater than levels observed in pento-blocked, OVX, E-primed rats treated with saline pulses from 1500–1800 h (P < 0.001). Coadministration of galanin at either 5 or 10 µg/pulse resulted in a significant potentiation of GnRH-stimulated LH secretion in OVX, E-primed animals. Animals treated with GnRH + 5 µg galanin/pulse exhibited a mean plasma LH level of 36.32 ± 5.07 ng/ml between 1500 and 1800 h, a mean level that was 1.3-fold greater than levels observed in response to treatment with the same dose of GnRH alone (P < 0.05). Likewise, coadministration of GnRH +10 µg galanin/pulse resulted in a mean plasma LH level of 39.11 ± 3.34 ng/ml between 1500 and 1800 h, a mean level that was 1.4-fold greater than levels observed in animals treated with the same dose of GnRH alone (P < 0.005; see Fig. 2C).
Effects of pento on LH secretion in OVX, E+P-treated ratsy3y+, 百拇医药
OVX, E-primed rats treated with P at 1100 h, an ip injection of saline at 1330 h, and pulses of saline from 1400–1730 h exhibited an LH surge on the afternoon of experiments. The mean LH level in OVX, E+P-treated control animals was 12.56 ± 3.05 ng/ml between 1500 and 1800 h (n = 6; see Fig. 3A). Administration of an ip injection of pento at 1330 h effectively blocked the steroid-induced LH surge in OVX, E+P-treated animals; mean plasma LH levels in these animals were 0.82 ± 0.22 ng/ml from 1500–1800 h, a mean level that was significantly lower than that observed in OVX, E+P-treated controls (P < 0.05).y3y+, 百拇医药
fig.ommitteedfig.ommitteedy3y+, 百拇医药
Figure 3. A, Effects of ip administration of pento (40 mg/kg BW) on plasma LH levels in OVX, E+P-treated rats. B, Effects of iv galanin administration (5 or 10 µg/pulse) on plasma LH levels in OVX, E+P-treated, pento-blocked rats. C, Effects of coadministration of galanin (5 or 10 µg/pulse) and GnRH (25 ng/pulse) on plasma LH levels in OVX, E+P-treated, pento-blocked rats. Note the difference in scale of the y-axis in C.
Effects of galanin alone on LH release in pento-blocked, OVX, E+P-treated ratso/i83i@, 百拇医药
Pulsatile administration of galanin alone at either dose used in the present study had no effect on LH secretion in OVX, E+P-treated rats. The mean LH level in OVX, E+P-treated rats treated with a 5 µg/pulse of galanin from 1400–1730 h was 1.47 ± 0.25 ng/ml; this mean LH level did not differ significantly from that observed in OVX, E+P-treated, pento-blocked rats that received pulses of saline alone from 1400–1730 h. Likewise, treatment of OVX, E+P-treated rats with 10 µg/pulse of galanin resulted in a mean LH level of 0.77 ± 0.12 ng/ml, a mean LH level that did not differ significantly from levels observed in OVX, E+P-treated, pento-blocked control animals (see Fig. 3B).o/i83i@, 百拇医药
Effects of galanin on GnRH-stimulated LH secretion in pento-blocked, OVX, E+P-treated ratso/i83i@, 百拇医药
Pulsatile administration of GnRH at a dose of 25 ng/pulse to pento-blocked, OVX, E+P-treated rats caused a significant elevation in mean plasma LH levels when compared with LH levels in pento-blocked, saline-treated control rats within the same steroid treatment group (29.07 ± 3.82 ng/ml; n = 6; P < 0.001; see Fig. 3C. Coadministration of either dose of galanin with 25 ng/pulse of GnRH caused a significant potentiation of GnRH-stimulated LH secretion in OVX, E+P-treated rats. Combined infusions of galanin at a dose of 5 µg/pulse and GnRH at a dose of 25 ng/pulse resulted in a mean plasma LH level of 40.58 ± 7.08 ng/ml between 1500 and 1800 h, a level that was 1.4-fold greater than levels observed in OVX, E+P-treated animals that received the same dose of GnRH alone (P < 0.05). Combined infusions of galanin at a dose of 10 µg/pulse along with GnRH at a dose of 25 ng/pulse resulted in a mean plasma LH level of 40.75 ± 5.49 ng/ml between 1500 and 1800 h, a mean level that was also 1.4-fold greater than that observed in animals treated with the same dose of GnRH alone (P < 0.05; see Fig. 3C).
Discussiong, 百拇医药
A variety of experimental evidence now supports the notion that hypothalamic galanin neurons participate in the induction of preovulatory LH surges in female rats. During the female rat estrous cycle, galanin mRNA expression in GnRH neurons (13) and galanin content in the basal hypothalamus (8) are highest on the day of proestrus in female rats, indicating an activation of hypothalamic galanin systems within the context of the preovulatory endocrine milieu. Elevated levels of estrogen on the day of proestrus are thought to be responsible for the up-regulation of galanin production at this time, given that estrogen has been shown to induce galanin mRNA and protein expression in hypothalamic neurons under a variety of experimental circumstances (18, 19, 20). Increased hypothalamic galanin production likely contributes to the generation of preovulatory LH surges at both the hypothalamic and pituitary levels on the day of proestrus, by participating in the generation of the GnRH surge and enhancing GnRH-stimulated LH secretion, respectively. These hypotheses are based on the findings that both central and peripheral blockade of galanin’s actions attenuates steroid-induced LH surges in female rats (7).
The results of the present study provide evidence that the pituitary actions of galanin are enhanced under the endocrine conditions in which preovulatory LH surges are generated. Galanin was found to significantly enhance GnRH-stimulated LH secretion within the context of the preovulatory steroidal milieu; coadministration of galanin along with GnRH produced LH surges in steroid-primed animals that were 30–40% greater than LH surges observed in animals treated with the same dose of GnRH alone. This effect of galanin was found to be strictly modulatory in nature, in that pulsatile administration of galanin alone had no effect on LH secretion in steroid-primed animals. Comparisons of the peptide’s neuromodulatory effects across steroid treatment groups in the present study indicate that estrogen is responsible for up-regulating pituitary sensitivity to galanin, because galanin enhanced GnRH-stimulated LH secretion equally in OVX, E-primed and OVX, E+P-treated rats but had no potentiating effect in OVX, unprimed animals. These results suggest that, as galanin production within hypothalamic neurons increases on proestrus, pituitary sensitivity to the neuropeptide is also up-regulated—just as it is for other neuropeptides involved in the generation of LH surges (GnRH, neuropeptide Y; 14–16)—thereby ensuring a robust LH surge and ultimately ovulation.
Interestingly, our laboratory recently demonstrated that galanin also acts to potentiate GnRH-stimulated LH secretion in male rats; and, like the effects observed in female rats in the present study, the pituitary effects of galanin in males are steroid-dependent (21). It is likely that the GALR2 receptor subtype mediates the modulatory effects of galanin on GnRH-stimulated LH secretion in males, given that combined in situ hybridization immunohistochemistry studies have demonstrated the presence of GALR2 mRNA in pituitary gonadotropes (22). Whether the GALR2 receptor is expressed in gonadotropes of the female pituitary gland and mediates galanin’s effects on GnRH-stimulated LH secretion in females remains to be determined; however, it is interesting to note that the galanin receptor antagonist galantide, which attenuates steroid-induced LH surges when administered peripherally to OVX rats, is known to bind to the rat GALR2 receptor (23).h?34, http://www.100md.com
The cellular mechanism(s) whereby galanin enhances pituitary sensitivity to GnRH in steroid-primed animals is also unknown. Galanin may induce changes in GnRH receptor number and/or affinity or may augment the intracellular signal transduction mechanisms coupled to the GnRH receptor. There is evidence to suggest that galanin can modulate GnRH binding to anterior pituitary membranes obtained from chronically OVX rats (24). However, the effects observed were modest and occurred in response to treatment with a pharmacological dose of the peptide. Further studies will be necessary to determine the extent to which galanin can enhance GnRH binding to gonadotropes exposed to proestrous levels of estrogen. Because the galanin receptor subtype expressed on gonadotropes in female rats remains largely uncharacterized at this point in time, discussion of whether galanin receptor activation augments the signal transduction pathways mediating GnRH-stimulated LH secretion remains purely speculative. However, it is interesting to note that the GALR2 receptor, which is expressed in gonadotropes in the male (22), is capable of stimulating inositol phospholipid turnover, mobilizing intracellular calcium (25, 26), and stimulating MAPK activity (25), and all of these signal transduction mechanisms are known to be activated by the GnRH receptor in pituitary gonadotropes (27, 28, 29). It is noteworthy that the potentiating effects of galanin on GnRH-stimulated LH secretion in both OVX, E-primed and OVX, E+P-treated animals were not evident until 1600 h on the afternoon of experiments in the present study. Given that pulsatile hormone administration was initiated at 1400 h, this observation would suggest that there is a 2-h lag in time between exposure to galanin and the onset of the peptide’s potentiating effect on GnRH-stimulated LH secretion.
The ability of galanin to potentiate GnRH-stimulated LH secretion in OVX, E-primed and OVX, E+P-treated animals was not found to be dose-dependent in the present study. In these two steroid treatment groups, mean LH levels did not differ significantly between animals treated with GnRH + 5 µg/pulse galanin and GnRH +10 µg/pulse galanin. A likely explanation for this lack of dose-dependence may be that, on coadministration of the lower dose of galanin along with GnRH, gonadotropes are responding maximally to stimulation by the neuropeptides, and thus, administration of a higher dose of galanin along with GnRH produces no further increase in LH secretion. It is noteworthy, in the present study, that P treatment resulted in no further enhancement of galanin’s effects on GnRH-stimulated LH secretion in estrogen-primed, OVX rats. This result contradicts the findings of Sanchez-Criado et al. (30), who observed that the antiprogestin RU486 blunts the stimulatory effects of galanin on GnRH-stimulated LH secretion from proestrous pituitaries in vitro. Although P treatment did not further enhance galanin’s effects on GnRH-stimulated LH secretion in estrogen-primed, OVX rats in the present study, it did shift the time course of the GnRH-induced LH surge, in that the peak of the surge occurred at 1500 h in animals treated with GnRH alone vs. 1600 h in animals treated with GnRH + 5 or 10 µg/pulse of galanin. It is anticipated that further experiments regarding the intracellular mechanism(s) whereby galanin enhances GnRH-stimulated LH secretion, and the impact of gonadal steroids on galanin receptor-induced signal transduction in gonadotropes, will reveal the significance of this phenomenon.
Interestingly, not only were the potentiating effects of galanin absent in OVX rats in the present study, but the peptide was actually found to inhibit GnRH-stimulated LH secretion in the complete absence of gonadal steroids. A similar inhibitory effect of galanin on GnRH-stimulated LH secretion has been observed in castrated male rats (21) and in anterior pituitaries harvested from proestrus female rats and cultured in the absence of estrogen (31). Taken together, results from these studies suggest that removal of gonadal steroid feedback unmasks an inhibitory effect of galanin on GnRH-stimulated LH secretion. Further in vivo studies will be necessary to determine the physiological significance of this inhibitory phenomenon, and to examine whether it occurs only in the complete absence of estrogen (i.e. in gonadectomized animals), or whether it exists in females at times during the estrous cycle when circulating levels of estrogen are low (i.e. in the presence of negative feedback levels of estrogen). Intriguingly, Cardenas et al. (32) observed, in their in vitro studies, that galanin is capable of potentiating GnRH-stimulated LH secretion from hemipituitary fragments obtained from diestrus 1 rats. This finding would suggest that the potentiating effects of galanin are maintained in the presence of low levels of estrogen, and that the inhibitory effects of the peptide, if present under physiological circumstances, are limited to conditions of hypogonadism.
In addition to hypothalamic neurons serving as a source of galanin, immunocytochemical studies have demonstrated that the anterior pituitary itself synthesizes a significant quantity of galanin (33). Immunoreactive galanin in the pituitary gland is mainly localized to lactotropes (34), and studies in female rats have demonstrated that estrogen is a potent stimulator of both galanin mRNA expression and protein synthesis in lactotropes (33, 35). Given that lactotropes exist within close proximity to gonadotropes within the dorsocephalic region of the anterior pituitary gland (36, 37), it is likely that lactotropes (in addition to hypothalamic galanin neurons) serve as a significant source of galanin at times of estrous cycle when estrogen levels are high (i.e. during the preovulatory period). Pituitary galanin may then act in a paracrine manner to enhance pituitary sensitivity to GnRH at the time of the preovulatory LH surge.h'[m', http://www.100md.com
It is now widely recognized that at the time of the preovulatory LH surge, the anterior pituitary gland is 50 times more sensitive to GnRH stimulation than it is during the rest of the estrous cycle (14). This acute increase in pituitary responsiveness to GnRH stimulation has been attributed to a number of factors, including GnRH self-priming (38), the direct effects of adrenal or ovarian P on the pituitary gland (39, 40), and the priming actions of neuropeptide Y (41). The results from the present study indicate that the neuropeptide galanin should also be added to this growing list of factors that act to ensure the efficacy of the GnRH—and hence, the LH—surge, and ultimately ovulation.
In conclusion, we have demonstrated that galanin significantly enhances GnRH-stimulated LH secretion in the presence of high levels of estrogen (in OVX, E-primed and OVX, E+P-treated animals). Galanin does not potentiate GnRH-stimulated LH secretion in OVX, unprimed animals; and, in fact, the neuropeptide inhibits GnRH-stimulated LH secretion in the absence of gonadal steroids. Administration of galanin alone (without GnRH) has no effect on LH secretion under any of the steroid treatment conditions examined in the present study. These results strongly implicate a role for galanin in the up-regulation of pituitary sensitivity to GnRH at the time of the preovulatory LH surge in female rats. Further studies are necessary to delineate the cellular mechanism(s) whereby galanin exerts its modulatory effects in pituitary gonadotropes.eceived August 15, 2002.;7, 百拇医药
Accepted for publication October 15, 2002.;7, 百拇医药
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