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Nitric oxide mediates leptin-induced preovulatory luteinizing hormone and prolactin surges in rats
Brain Research 923 (2001) 193–197 www.elsevier.com / locate / bres
Short communication
Nitric oxide mediates leptin-induced preovulatory luteinizing hormone and prolactin surges in rats ¨ b Hajime Watanobe a , *, Helgi B. Schioth a
Division of Internal Medicine, Clinical Research Center, International University of Health and Welfare, 2600 -1 Kitakanemaru, Otawara, Tochigi 324 -8501, Japan b Department of Neuroscience, Uppsala University, BMC, Box 593, 751 24 Uppsala, Sweden Accepted 5 October 2001
Abstract The objective of this study was to investigate whether nitric oxide (NO) plays a significant role in mediating the facilitatory action of leptin on the reproductive system. The covariate of reproductive function we used for evaluation was preovulatory surges of luteinizing hormone (LH) and prolactin (PRL), which were simulated by priming ovariectomized rats with estradiol and progesterone. A systemic treatment of normally-fed rats with an NO synthase inhibitor (N W-nitro-L-arginine methyl ester) significantly decreased the magnitude of both LH and PRL surges. Three-day-fasted rats did not show a significant surge of either LH or PRL. An intracerebroventricular administration of leptin to fasted rats led to a significant recovery of these hormonal surges, but a simultaneous administration of both the NO synthase inhibitor and leptin significantly abrogated the effects of leptin. This is the first report to demonstrate a significant intermediary role of NO in leptin-induced preovulatory LH and PRL surges in rats. 2001 Elsevier Science B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Hypothalamic–pituitary–gonadal regulation Keywords: Nitric oxide; Leptin; Luteinizing hormone; Prolactin; Surge
Leptin is an adipocyte-derived hormone that regulates metabolic efficiency, energy expenditure, and food intake [12]. Besides its well-known role in body weight homeostasis, increasing evidence suggests that leptin may play a significant role in the regulation of neuroendocrine functions [2]. Leptin has been implicated in regulating the somatotropic, thyroid, adrenal, and reproductive axes. Among these, the proposed link between leptin and the reproductive system seems to be of utmost physiological significance in view of the established role for body fat, the source of leptin, in regulating the reproductive capability [2]. The preovulatory surge-like secretion of luteinizing hormone (LH) and prolactin (PRL) is a crucial physiological event in mammalian reproduction. We have previously reported that leptin exerts a facilitatory effect on these *Corresponding author. Tel. / fax: 181-287-24-1248. E-mail address: [email protected] (H. Watanobe).
hormonal surges, and this action of the adipose hormone may, at least in part, be mediated by the central melanocortin 4 receptor [25]. This significant involvement of the melanocortin 4 receptor in LH secretion in female rats was recently confirmed by Murray et al. [19]. Additional systems which were proposed to mediate the leptin stimulation of reproductive function comprise the cocaine- and amphetamine-regulated transcript [17,18,21], which is an endogenous inhibitor of food intake [16], prostaglandins [7], and serotonin neurons in the brainstem [9]. However, the neuroendocrine circuitry mediating leptin’s action on the hypothalamo–pituitary–gonadal axis is far from being fully elucidated. Nitric oxide (NO) is a gaseous neurotransmitter that is known to play important roles in a variety of physiological processes including the neuroendocrine functions [6]. As regards the hypothalamo–pituitary–gonadal axis, a good body of evidence suggests a stimulatory effect of NO on this endocrine axis [6]. In this connection, it is interesting
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )03247-4
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to note that Yu et al. [28] reported a significant intermediary role of NO in leptin-stimulated release of gonadotropin-releasing hormone (GnRH) and LH in vitro in the rat. However, we are not aware of any study that has further investigated the alleged interplay between leptin and NO in regulating reproductive function. In the present study, we thus examined whether NO plays a significant role in mediating the leptin-induced preovulatory LH and PRL surges in rats, which can be simulated by priming ovariectomized rats with estrogen and progesterone. All the following procedures were approved by the Ethical Committee for Animal Experiments of the International University of Health and Welfare. Animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Female rats (250–270 g) of the Wistar strain were used. They were housed in an air-conditioned room with controlled lighting (light 08:00–20:00 h), and were given free access to laboratory chow and tap water. The animals were ovariectomized under light ether anesthesia about 2 weeks before experimentation. Seven to 10 days before experiments, the animals were anesthetized with sodium pentobarbital (40 mg / kg body weight, intraperitoneally), and a guide cannula (22 gauge) with a removable inner stylet was stereotaxically implanted towards a lateral cerebroventricle. Coordinates for placement of the cannula were the same as in our previous reports [15,25,26]. The cannula was fixed onto the skull with anchor screws and dental cement. Two days prior to the experiment, the animals were implanted with a jugular vein catheter filled with heparin solution, and also implanted subcutaneously with a single Silastic capsule containing 300 mg / ml of estradiol17b (Sigma, St. Louis, MO, USA) under light ether anesthesia, in the same manner as in our previous studies [14,15,25,26]. Experiments were performed on both normally-fed (NF) and 3-day-fasted (F) rats. At about 8:00 h on the day of the experiment, the jugular vein catheter was exteriorized for frequent blood sampling. The inner stylet of the guide cannula was removed and replaced with a 30-gauge injection needle connected to Teflon tubing. At 9:00 h, 5 mg of progesterone (Mochida Pharmaceutical Co., Ltd., Tokyo, Japan) was injected intramuscularly into each rat. A total of five experimental groups were prepared. NF rats received a subcutaneous administration of 40 mg / kg of N W-nitro-L-arginine methyl ester (NAME; Sigma), an NO synthase (NOS) inhibitor, dissolved in physiological saline (SAL) at 10:00, 12:00, and 14:00 h. Control NF rats were injected SAL only at the same time points. These treatments were done in accordance with the method described by Bonavera et al. [3,4]. At 11:00 h, both groups received an intracerebroventricular administration of artificial cerebrospinal fluid (aCSF), which had the same composition as in our previous report [24]. These two subsets of NF animals were designated as NF1NAME1aCSF or NF1
SAL1aCSF group, respectively. The F rats were divided into three subsets. F1SAL1aCSF group were treated in the same manner as NF1SAL1aCSF group. F1SAL1 leptin group were administered SAL subcutaneously, and 0.3 nmol of recombinant rat leptin (R&D Systems, Minneapolis, MN, USA) dissolved in aCSF intracerebroventricularly. One more subset of F animals designated as F1NAME1leptin group received NAME and leptin in the same manner as NF1NAME1aCSF or F1SAL1leptin group, respectively. Every intracerebroventricular injection was done in a volume of 5 ml over 2–3 min. Blood samples (200 ml) were collected every 30 min over a total period of 420 min (11:00–18:00 h). To prevent the loss of circulating plasma volume, 200 ml of 0.9% NaCl was injected immediately after each blood collection. The blood was collected in EDTA-2Na (2.5 mg / ml)-containing tubes, centrifuged, and the plasma was stored at 2708C until assayed for LH and PRL. Within 30 min of the experiment, 15–20 ml of 0.1% methylene blue solution was injected intracerebroventricularly, and then the animals were sacrificed by decapitation. The brains were removed, and we checked whether the median eminence was stained with the dye. Only animals whose median eminence was dyed were considered to have undergone a successful intracerebroventricular injection, and allowed to contribute to the data given below. Plasma LH and PRL levels were determined by RIA using reagents kindly donated by Dr. A.F. Parlow (NIDDK). Rat LH-RP-3 and PRL-RP-3 were used as the standards. The sensitivity of the LH assay was 0.2 ng / ml, and that of PRL assay was 0.8 ng / ml. For both hormones, samples from individual rats were analyzed within the same assay. Both intra- and inter-assay coefficients of variation were less than 10% in the two assays. Results were expressed as the mean6S.E.M. One-way or two-way analysis of variance (ANOVA) followed by Scheffe’s post-hoc test was used to analyze the data. Differences were considered significant if P was smaller than 0.05. Fig. 1 shows the temporal profiles of plasma LH and PRL in the NF1SAL1aCSF, NF1NAME1aCSF, and F1SAL1aCSF groups. As regards LH levels (Fig. 1A), the NF1SAL1aCSF group showed significantly higher levels of the hormone (LH surge) during the period of 14:30–18:00 h than at 11:00 h. The NF1NAME1aCSF group also showed a significant LH surge during the period of 16:00–18:00 h as compared to their 11:00-h value. However, the magnitude of LH surge in this group was significantly smaller than that in the NF1SAL1aCSF group between 15:00 and 18:00 h. In agreement with our previous studies [15,25,26], the F1SAL1aCSF group did not show a significant LH surge. Plasma LH levels in the NF1NAME1aCSF group were significantly higher than those in the F1SAL1aCSF group during the period of 16:30–18:00 h. A similar finding was also observed for plasma PRL levels (Fig. 1B). Significantly higher levels of
¨ / Brain Research 923 (2001) 193 – 197 H. Watanobe, H.B. Schioth
Fig. 1. Effects of NOS inhibition on ovarian steroid-induced LH and PRL surges in NF ovariectomized rats. The number of rats examined was 10–12 per group. s–s, NF1SAL1aCSF; h–h, NF1NAME1aCSF; n–n, F1SAL1aCSF. †, Significantly different vs. the other two groups. *, Significantly different vs. F1SAL1aCSF group. In this and the next figures, arrows indicate the time points when NAME (40 mg / kg) or SAL was injected subcutaneously. aCSF was given intracerebroventricularly. In this and the next figures, where standard errors are not shown, they were smaller than the symbols. For further details, see text.
PRL than 11:00-h values (PRL surge) were observed in both the NF1SAL1aCSF and NF1NAME1aCSF groups during the period of 14:00–18:00 h or 15:30–18:00 h, respectively. Although the NF1NAME1aCSF group had significantly higher levels of PRL than the F1SAL1aCSF group between 16:30 and 18:00 h, the overall magnitude of PRL surge in the former group was significantly smaller than that in the NF1SAL1aCSF group. Fig. 2 shows the effects of NAME and leptin on LH and PRL surges in F animals. The data of the NF1SAL1aCSF and F1SAL1aCSF groups are shown again for comparison. The F1SAL1leptin group exhibited a significant recovery of LH surge (Fig. 2A). The magnitude of LH surge in this group was slightly smaller than that in the
195
Fig. 2. Effects of leptin and a combined administration of leptin and NAME on ovarian steroid-induced LH and PRL surges in F ovariectomized rats. The number of rats examined was 9–12 per group. s–s, NF1SAL1aCSF; h–h, F1SAL1leptin; d–d, F1NAME1leptin; n– n, F1SAL1aCSF. Leptin (0.3 nmol) and aCSF were given intracerebroventricularly. †, Significantly different vs. F1NAME1leptin and F1 SAL1aCSF groups. *, Significantly different vs. F1SAL1aCSF group. For further details, see text.
NF1SAL1aCSF group, but this difference was statistically insignificant. Interestingly, the F1NAME1leptin group had a significantly smaller magnitude of LH surge than the F1SAL1leptin group, although the LH levels in the former group still exceeded those in the F1SAL1aCSF group between 16:30 and 18:00 h. A very similar finding was observed for PRL surge (Fig. 2B). Leptin given to F rats significantly reinstated PRL surge (F1SAL1leptin group), but a combined administration of NAME and leptin led to a significant inhibition of the stimulatory effect of leptin (F1NAME1leptin group). Ample evidence is in favor of a significant role of NO in regulating the reproductive axis [6]. Several previous studies including our own have reported a significant contribution of NO to the preovulatory LH surge, which is
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a crucial physiological event in mammalian reproduction [1,3–5,14]. Pu et al. [22] reported that in both normallycycling and ovarian steroid-primed gonadectomized female rats, the extracellular efflux of cyclic GMP, a reliable index of NO activity, increases during the LH surge in the medial preoptic area, a hypothalamic site where the majority of GnRH neuronal cell bodies are located. We also demonstrated in a similar rat model that direct infusion of an NO donor into the medial preoptic area elevates the local release of cyclic GMP and GnRH in association with the advanced onset of the LH surge [14]. In the present study, we administered NAME in exactly the same manner as Bonavera et al. [3,4]. Our current data that this treatment resulted in a significant suppression of the preovulatory LH surge are in good agreement with what Bonavera et al. [3,4] reported. These investigators also found a stimulatory effect of NO on the preovulatory PRL surge, and this observation was confirmed by our present study. To date, there is only one study that tested whether NO is involved in leptin stimulation of reproductive function. Yu et al. [28] reported that NOS inhibition completely suppressed leptin-induced release of GnRH from rat hypothalamic explants in vitro, although its basal secretion was not affected by decreasing NO. They also found that LH release from rat anterior pituitaries induced by leptin in vitro was completely abrogated by NOS inhibition, but basal LH release was not. From these data, Yu et al. [28] concluded that leptin may act at both the hypothalamus and the pituitary to stimulate NO release, which then induces the release of either GnRH or LH, respectively. The current findings that food deprivation abolished, and leptin substitution reinstated the LH and PRL surges, are in good agreement with our previous studies [15,25,26]. In addition, the present study demonstrated for the first time that a combined administration of leptin and an NOS inhibitor led to a significant suppression of leptin’s facilitatory effects on LH and PRL surges. This finding can be construed as suggesting a significant intermediary role of NO in leptin stimulation of the preovulatory hormonal surges. Therefore, our present data support the general conclusion of Yu et al. [28], even though they and we employed distinct covariates to evaluate the reproductive function. In addition, the current study appears to be the first to demonstrate that the leptin-induced PRL surge is, at least partially, mediated by NO. Our data may be in agreement with previous studies by other investigators that leptin enhanced NOS activity in several hypothalamic nuclei in rats [11,20]. However, it was also reported that such changes were not observed in either neonatal rats [7] or mice [8]. It deserves attention that in the current study plasma LH and PRL levels in the F1NAME1leptin group were still significantly higher than those in the F1SAL1aCSF group. There are two possible explanations for this finding. Firstly, the dose of NAME we used might not have been high enough to completely block the endogenous pro-
duction of NO. Thus, a larger dose of NAME might have completely abolished the leptin-induced reinstatement of the preovulatory LH and PRL surges. Indeed, Yu et al. [28] reported that the inhibitory effect of NOS inhibition on leptin-stimulated release of GnRH and LH was complete. Secondly, it is also possible that other molecules than NO may significantly mediate the neuroendocrine effects of leptin. Such candidate systems comprise the melanocortin 4 receptor signaling [19,25], cocaine- and amphetamineregulated transcript [17,18,21], prostaglandins [7], and serotonin [9]. A significant role of NO has also been reported for other biological activities of leptin. It was suggested that vasorelaxant effects of the adipose hormone may be mediated by NO produced locally by vascular endothelial cells [10,13,27]. A significant mediation by NO of leptin’s stimulatory influence on glucose uptake in skeletal muscle was also reported [23]. In summary, in this study we demonstrated for the first time that NO significantly mediates leptin’s facilitatory action on the preovulatory LH and PRL surges in rats. Although there are additional candidates that may mediate the neuroendocrine effects of leptin, our present data suggest that NO may also play a significant role.
Acknowledgements We thank the National Hormone and Pituitary Program of NIDDK and Dr. A.F. Parlow for the generous donation of reagents for rat LH and PRL RIAs. This study was supported in part by a grant-in-aid from the Japan Society for the Promotion of Science (No. 12671072) to H.W. H.B.S. was supported by the Swedish Medical Research Council (MRC), the Swedish Society for Medical Re˚ search (SSMF), Ake Wibergs Stiftelse and Melacure Therapeutics AB, Uppsala, Sweden.
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Short communication
Nitric oxide mediates leptin-induced preovulatory luteinizing hormone and prolactin surges in rats ¨ b Hajime Watanobe a , *, Helgi B. Schioth a
Division of Internal Medicine, Clinical Research Center, International University of Health and Welfare, 2600 -1 Kitakanemaru, Otawara, Tochigi 324 -8501, Japan b Department of Neuroscience, Uppsala University, BMC, Box 593, 751 24 Uppsala, Sweden Accepted 5 October 2001
Abstract The objective of this study was to investigate whether nitric oxide (NO) plays a significant role in mediating the facilitatory action of leptin on the reproductive system. The covariate of reproductive function we used for evaluation was preovulatory surges of luteinizing hormone (LH) and prolactin (PRL), which were simulated by priming ovariectomized rats with estradiol and progesterone. A systemic treatment of normally-fed rats with an NO synthase inhibitor (N W-nitro-L-arginine methyl ester) significantly decreased the magnitude of both LH and PRL surges. Three-day-fasted rats did not show a significant surge of either LH or PRL. An intracerebroventricular administration of leptin to fasted rats led to a significant recovery of these hormonal surges, but a simultaneous administration of both the NO synthase inhibitor and leptin significantly abrogated the effects of leptin. This is the first report to demonstrate a significant intermediary role of NO in leptin-induced preovulatory LH and PRL surges in rats. 2001 Elsevier Science B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Hypothalamic–pituitary–gonadal regulation Keywords: Nitric oxide; Leptin; Luteinizing hormone; Prolactin; Surge
Leptin is an adipocyte-derived hormone that regulates metabolic efficiency, energy expenditure, and food intake [12]. Besides its well-known role in body weight homeostasis, increasing evidence suggests that leptin may play a significant role in the regulation of neuroendocrine functions [2]. Leptin has been implicated in regulating the somatotropic, thyroid, adrenal, and reproductive axes. Among these, the proposed link between leptin and the reproductive system seems to be of utmost physiological significance in view of the established role for body fat, the source of leptin, in regulating the reproductive capability [2]. The preovulatory surge-like secretion of luteinizing hormone (LH) and prolactin (PRL) is a crucial physiological event in mammalian reproduction. We have previously reported that leptin exerts a facilitatory effect on these *Corresponding author. Tel. / fax: 181-287-24-1248. E-mail address: [email protected] (H. Watanobe).
hormonal surges, and this action of the adipose hormone may, at least in part, be mediated by the central melanocortin 4 receptor [25]. This significant involvement of the melanocortin 4 receptor in LH secretion in female rats was recently confirmed by Murray et al. [19]. Additional systems which were proposed to mediate the leptin stimulation of reproductive function comprise the cocaine- and amphetamine-regulated transcript [17,18,21], which is an endogenous inhibitor of food intake [16], prostaglandins [7], and serotonin neurons in the brainstem [9]. However, the neuroendocrine circuitry mediating leptin’s action on the hypothalamo–pituitary–gonadal axis is far from being fully elucidated. Nitric oxide (NO) is a gaseous neurotransmitter that is known to play important roles in a variety of physiological processes including the neuroendocrine functions [6]. As regards the hypothalamo–pituitary–gonadal axis, a good body of evidence suggests a stimulatory effect of NO on this endocrine axis [6]. In this connection, it is interesting
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )03247-4
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to note that Yu et al. [28] reported a significant intermediary role of NO in leptin-stimulated release of gonadotropin-releasing hormone (GnRH) and LH in vitro in the rat. However, we are not aware of any study that has further investigated the alleged interplay between leptin and NO in regulating reproductive function. In the present study, we thus examined whether NO plays a significant role in mediating the leptin-induced preovulatory LH and PRL surges in rats, which can be simulated by priming ovariectomized rats with estrogen and progesterone. All the following procedures were approved by the Ethical Committee for Animal Experiments of the International University of Health and Welfare. Animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Female rats (250–270 g) of the Wistar strain were used. They were housed in an air-conditioned room with controlled lighting (light 08:00–20:00 h), and were given free access to laboratory chow and tap water. The animals were ovariectomized under light ether anesthesia about 2 weeks before experimentation. Seven to 10 days before experiments, the animals were anesthetized with sodium pentobarbital (40 mg / kg body weight, intraperitoneally), and a guide cannula (22 gauge) with a removable inner stylet was stereotaxically implanted towards a lateral cerebroventricle. Coordinates for placement of the cannula were the same as in our previous reports [15,25,26]. The cannula was fixed onto the skull with anchor screws and dental cement. Two days prior to the experiment, the animals were implanted with a jugular vein catheter filled with heparin solution, and also implanted subcutaneously with a single Silastic capsule containing 300 mg / ml of estradiol17b (Sigma, St. Louis, MO, USA) under light ether anesthesia, in the same manner as in our previous studies [14,15,25,26]. Experiments were performed on both normally-fed (NF) and 3-day-fasted (F) rats. At about 8:00 h on the day of the experiment, the jugular vein catheter was exteriorized for frequent blood sampling. The inner stylet of the guide cannula was removed and replaced with a 30-gauge injection needle connected to Teflon tubing. At 9:00 h, 5 mg of progesterone (Mochida Pharmaceutical Co., Ltd., Tokyo, Japan) was injected intramuscularly into each rat. A total of five experimental groups were prepared. NF rats received a subcutaneous administration of 40 mg / kg of N W-nitro-L-arginine methyl ester (NAME; Sigma), an NO synthase (NOS) inhibitor, dissolved in physiological saline (SAL) at 10:00, 12:00, and 14:00 h. Control NF rats were injected SAL only at the same time points. These treatments were done in accordance with the method described by Bonavera et al. [3,4]. At 11:00 h, both groups received an intracerebroventricular administration of artificial cerebrospinal fluid (aCSF), which had the same composition as in our previous report [24]. These two subsets of NF animals were designated as NF1NAME1aCSF or NF1
SAL1aCSF group, respectively. The F rats were divided into three subsets. F1SAL1aCSF group were treated in the same manner as NF1SAL1aCSF group. F1SAL1 leptin group were administered SAL subcutaneously, and 0.3 nmol of recombinant rat leptin (R&D Systems, Minneapolis, MN, USA) dissolved in aCSF intracerebroventricularly. One more subset of F animals designated as F1NAME1leptin group received NAME and leptin in the same manner as NF1NAME1aCSF or F1SAL1leptin group, respectively. Every intracerebroventricular injection was done in a volume of 5 ml over 2–3 min. Blood samples (200 ml) were collected every 30 min over a total period of 420 min (11:00–18:00 h). To prevent the loss of circulating plasma volume, 200 ml of 0.9% NaCl was injected immediately after each blood collection. The blood was collected in EDTA-2Na (2.5 mg / ml)-containing tubes, centrifuged, and the plasma was stored at 2708C until assayed for LH and PRL. Within 30 min of the experiment, 15–20 ml of 0.1% methylene blue solution was injected intracerebroventricularly, and then the animals were sacrificed by decapitation. The brains were removed, and we checked whether the median eminence was stained with the dye. Only animals whose median eminence was dyed were considered to have undergone a successful intracerebroventricular injection, and allowed to contribute to the data given below. Plasma LH and PRL levels were determined by RIA using reagents kindly donated by Dr. A.F. Parlow (NIDDK). Rat LH-RP-3 and PRL-RP-3 were used as the standards. The sensitivity of the LH assay was 0.2 ng / ml, and that of PRL assay was 0.8 ng / ml. For both hormones, samples from individual rats were analyzed within the same assay. Both intra- and inter-assay coefficients of variation were less than 10% in the two assays. Results were expressed as the mean6S.E.M. One-way or two-way analysis of variance (ANOVA) followed by Scheffe’s post-hoc test was used to analyze the data. Differences were considered significant if P was smaller than 0.05. Fig. 1 shows the temporal profiles of plasma LH and PRL in the NF1SAL1aCSF, NF1NAME1aCSF, and F1SAL1aCSF groups. As regards LH levels (Fig. 1A), the NF1SAL1aCSF group showed significantly higher levels of the hormone (LH surge) during the period of 14:30–18:00 h than at 11:00 h. The NF1NAME1aCSF group also showed a significant LH surge during the period of 16:00–18:00 h as compared to their 11:00-h value. However, the magnitude of LH surge in this group was significantly smaller than that in the NF1SAL1aCSF group between 15:00 and 18:00 h. In agreement with our previous studies [15,25,26], the F1SAL1aCSF group did not show a significant LH surge. Plasma LH levels in the NF1NAME1aCSF group were significantly higher than those in the F1SAL1aCSF group during the period of 16:30–18:00 h. A similar finding was also observed for plasma PRL levels (Fig. 1B). Significantly higher levels of
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Fig. 1. Effects of NOS inhibition on ovarian steroid-induced LH and PRL surges in NF ovariectomized rats. The number of rats examined was 10–12 per group. s–s, NF1SAL1aCSF; h–h, NF1NAME1aCSF; n–n, F1SAL1aCSF. †, Significantly different vs. the other two groups. *, Significantly different vs. F1SAL1aCSF group. In this and the next figures, arrows indicate the time points when NAME (40 mg / kg) or SAL was injected subcutaneously. aCSF was given intracerebroventricularly. In this and the next figures, where standard errors are not shown, they were smaller than the symbols. For further details, see text.
PRL than 11:00-h values (PRL surge) were observed in both the NF1SAL1aCSF and NF1NAME1aCSF groups during the period of 14:00–18:00 h or 15:30–18:00 h, respectively. Although the NF1NAME1aCSF group had significantly higher levels of PRL than the F1SAL1aCSF group between 16:30 and 18:00 h, the overall magnitude of PRL surge in the former group was significantly smaller than that in the NF1SAL1aCSF group. Fig. 2 shows the effects of NAME and leptin on LH and PRL surges in F animals. The data of the NF1SAL1aCSF and F1SAL1aCSF groups are shown again for comparison. The F1SAL1leptin group exhibited a significant recovery of LH surge (Fig. 2A). The magnitude of LH surge in this group was slightly smaller than that in the
195
Fig. 2. Effects of leptin and a combined administration of leptin and NAME on ovarian steroid-induced LH and PRL surges in F ovariectomized rats. The number of rats examined was 9–12 per group. s–s, NF1SAL1aCSF; h–h, F1SAL1leptin; d–d, F1NAME1leptin; n– n, F1SAL1aCSF. Leptin (0.3 nmol) and aCSF were given intracerebroventricularly. †, Significantly different vs. F1NAME1leptin and F1 SAL1aCSF groups. *, Significantly different vs. F1SAL1aCSF group. For further details, see text.
NF1SAL1aCSF group, but this difference was statistically insignificant. Interestingly, the F1NAME1leptin group had a significantly smaller magnitude of LH surge than the F1SAL1leptin group, although the LH levels in the former group still exceeded those in the F1SAL1aCSF group between 16:30 and 18:00 h. A very similar finding was observed for PRL surge (Fig. 2B). Leptin given to F rats significantly reinstated PRL surge (F1SAL1leptin group), but a combined administration of NAME and leptin led to a significant inhibition of the stimulatory effect of leptin (F1NAME1leptin group). Ample evidence is in favor of a significant role of NO in regulating the reproductive axis [6]. Several previous studies including our own have reported a significant contribution of NO to the preovulatory LH surge, which is
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a crucial physiological event in mammalian reproduction [1,3–5,14]. Pu et al. [22] reported that in both normallycycling and ovarian steroid-primed gonadectomized female rats, the extracellular efflux of cyclic GMP, a reliable index of NO activity, increases during the LH surge in the medial preoptic area, a hypothalamic site where the majority of GnRH neuronal cell bodies are located. We also demonstrated in a similar rat model that direct infusion of an NO donor into the medial preoptic area elevates the local release of cyclic GMP and GnRH in association with the advanced onset of the LH surge [14]. In the present study, we administered NAME in exactly the same manner as Bonavera et al. [3,4]. Our current data that this treatment resulted in a significant suppression of the preovulatory LH surge are in good agreement with what Bonavera et al. [3,4] reported. These investigators also found a stimulatory effect of NO on the preovulatory PRL surge, and this observation was confirmed by our present study. To date, there is only one study that tested whether NO is involved in leptin stimulation of reproductive function. Yu et al. [28] reported that NOS inhibition completely suppressed leptin-induced release of GnRH from rat hypothalamic explants in vitro, although its basal secretion was not affected by decreasing NO. They also found that LH release from rat anterior pituitaries induced by leptin in vitro was completely abrogated by NOS inhibition, but basal LH release was not. From these data, Yu et al. [28] concluded that leptin may act at both the hypothalamus and the pituitary to stimulate NO release, which then induces the release of either GnRH or LH, respectively. The current findings that food deprivation abolished, and leptin substitution reinstated the LH and PRL surges, are in good agreement with our previous studies [15,25,26]. In addition, the present study demonstrated for the first time that a combined administration of leptin and an NOS inhibitor led to a significant suppression of leptin’s facilitatory effects on LH and PRL surges. This finding can be construed as suggesting a significant intermediary role of NO in leptin stimulation of the preovulatory hormonal surges. Therefore, our present data support the general conclusion of Yu et al. [28], even though they and we employed distinct covariates to evaluate the reproductive function. In addition, the current study appears to be the first to demonstrate that the leptin-induced PRL surge is, at least partially, mediated by NO. Our data may be in agreement with previous studies by other investigators that leptin enhanced NOS activity in several hypothalamic nuclei in rats [11,20]. However, it was also reported that such changes were not observed in either neonatal rats [7] or mice [8]. It deserves attention that in the current study plasma LH and PRL levels in the F1NAME1leptin group were still significantly higher than those in the F1SAL1aCSF group. There are two possible explanations for this finding. Firstly, the dose of NAME we used might not have been high enough to completely block the endogenous pro-
duction of NO. Thus, a larger dose of NAME might have completely abolished the leptin-induced reinstatement of the preovulatory LH and PRL surges. Indeed, Yu et al. [28] reported that the inhibitory effect of NOS inhibition on leptin-stimulated release of GnRH and LH was complete. Secondly, it is also possible that other molecules than NO may significantly mediate the neuroendocrine effects of leptin. Such candidate systems comprise the melanocortin 4 receptor signaling [19,25], cocaine- and amphetamineregulated transcript [17,18,21], prostaglandins [7], and serotonin [9]. A significant role of NO has also been reported for other biological activities of leptin. It was suggested that vasorelaxant effects of the adipose hormone may be mediated by NO produced locally by vascular endothelial cells [10,13,27]. A significant mediation by NO of leptin’s stimulatory influence on glucose uptake in skeletal muscle was also reported [23]. In summary, in this study we demonstrated for the first time that NO significantly mediates leptin’s facilitatory action on the preovulatory LH and PRL surges in rats. Although there are additional candidates that may mediate the neuroendocrine effects of leptin, our present data suggest that NO may also play a significant role.
Acknowledgements We thank the National Hormone and Pituitary Program of NIDDK and Dr. A.F. Parlow for the generous donation of reagents for rat LH and PRL RIAs. This study was supported in part by a grant-in-aid from the Japan Society for the Promotion of Science (No. 12671072) to H.W. H.B.S. was supported by the Swedish Medical Research Council (MRC), the Swedish Society for Medical Re˚ search (SSMF), Ake Wibergs Stiftelse and Melacure Therapeutics AB, Uppsala, Sweden.
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