Obesity and endocrine dysfunction in mice with deletions of both neuropeptide Y and galanin.

Neuropeptide Y (NPY) and galanin have both been implicated in the regulation of body weight, yet mice bearing deletions of either of these molecules have unremarkable metabolic phenotypes. To investigate whether galanin and NPY might compensate for one another, we produced mutants lacking both neuropeptides (GAL(-/-)/NPY(-/-)). We found that male GAL(-/-)/NPY(-/-) mice ate significantly more and were much heavier (30%) than wild-type (WT) controls. GAL(-/-)/NPY(-/-) mice responded to a high-fat diet by gaining more weight than WT mice gain, and they were unable to regulate their weight normally after a change in diet. GAL(-/-)/NPY(-/-) mice had elevated levels of leptin, insulin, and glucose, and they lost more weight than WT mice during chronic leptin treatment. Galanin mRNA was increased in the hypothalamus of NPY(-/-) mice, providing evidence of compensatory regulation in single mutants. The disruption of energy balance observed in GAL(-/-)/NPY(-/-) double knockouts is not found in the phenotype of single knockouts of either molecule. The unexpected obesity phenotype may result from the dysregulation of the leptin and insulin systems that normally keep body weight within the homeostatic range.

Kisspepeptin-GPR54 signaling in the neuroendocrine reproductive axis.

Kisspeptins, which are products of the Kiss1 gene, and their receptor, GPR54, have emerged as key players in the regulation of gonadotropin-releasing hormone (GnRH) secretion. Mutations or targeted deletions of GPR54 produce isolated hypogonadotropic hypogonadism in humans and mice, indicating that signaling through this receptor is a prerequisite for sexual maturation. Centrally administered kisspeptins stimulate GnRH and gonadotropin secretion in prepubertal and adult animals. Kisspeptin-expressing neurons are direct targets for the negative and positive feedback actions of sex steroids, which differentially regulate the expression of KiSS-1 mRNA in various regions of the forebrain. This review highlights what is currently known about kisspeptin-GPR54 signaling in the regulation of the neuroendocrine reproductive axis.

KiSS-1 neurones are direct targets for leptin in the ob/ob mouse.

Leptin is an adipocyte-derived hormone that acts on the hypothalamus to influence feeding, metabolism and reproduction, but the cellular and molecular targets for the action of leptin in the brain have yet to be fully elucidated. Kisspeptins are encoded by the Kiss1 gene, which is expressed in the hypothalamus and has been implicated in the neuroendocrine regulation of gonadotrophin-releasing hormone secretion. We tested the hypothesis that kisspeptin-expressing neurones are targets for leptin. First, we examined whether leptin regulates the expression of Kiss1 by comparing levels of KiSS-1 mRNA in the arcuate nucleus among groups of mice having different circulating levels of leptin: (i) wild-type (WT); (ii) leptin-deficient ob/ob; and (iii) ob/ob mice treated with leptin. All mice were castrated to control for endogenous concentrations of gonadal steroids. KiSS-1 mRNA was significantly reduced in ob/ob compared to WT mice and levels of KiSS-1 mRNA in ob/ob mice treated with leptin were increased, but not fully restored to that found in WT animals. Second, we performed double-label in situ hybridisation for KiSS-1 mRNA and the leptin receptor (Ob-Rb) mRNA and found that almost one-half (approximately 40%) of KiSS-1 mRNA-expressing cells in the arcuate nucleus expressed Ob-Rb mRNA. These results demonstrate that KiSS-1 neurones are direct targets for regulation by leptin and suggest that the reproductive deficits associated with leptin-deficient states may be attributable, in part, to diminished expression of Kiss1.

Galanin: an unassuming neuropeptide moves to center stage in reproduction.

Galanin is a neuropeptide widely distributed throughout the reproductive system, including the brain, pituitary, testes, ovaries, and accessory reproductive tracts of both sexes. Within the hypothalamus, a subset of GnRH-producing neurons also synthesizes galanin. Galanin gene expression in these GnRH neurons is highly inducible by estrogen. Galanin is thought to play an important role in the generation of the preovulatory LH surge, and may serve other functions yet to be discovered.

Neuroanatomical localization of cells containing gonadotropin-releasing hormone messenger ribonucleic acid in the primate brain by in situ hybridization histochemistry.

Using in situ hybridization histochemistry, we have mapped the anatomic localization of perikarya containing mRNA that codes for GnRH and GnRH-associated protein (GAP) in the forebrain of four male macaques, Macaca fascicularis. DNA oligomers, with sequences complementary to either the GnRH or the GAP portion of the mRNA sequence, were synthesized and hybridized to paraformaldehyde fixed, coronal sections of the basal forebrain and hypothalamus. GnRH mRNA was found in the same population of cells as those containing GAP mRNA. GnRH/GAP mRNA-containing cell bodies were observed consistently in the medial septal nucleus, the diagonal band of Broca, the medial preoptic area, supraoptic nucleus, and ventromedial-infundibular region. We detected the presence of GnRH mRNA and GAP mRNA within the same neuroanatomic regions previously shown to include perikarya containing immunoreactive GnRH. The ventromedial-infundibular region and the medial preoptic region contained the greatest number of GnRH/GAP mRNA-containing perikarya (37.0% and 22.5%, respectively). The diagonal band contained 21.0% and the supraoptic nucleus 13.0% of the cells, while the medial septum contained the fewest number (6.7%). This study demonstrates the feasibility of using in situ hybridization as a strategy to study the developmental and steroidal regulation of GnRH gene expression in the nonhuman primate.

Cycle detection: a technique for estimating the frequency and amplitude of episodic fluctuations in blood hormone and substrate concentrations.

Investigation of episodic endocrine secretion has been hampered by inadequate analytical techniques for describing patterns of blood concentrations over time. Although standard time series methods, such as autocorrelation and power spectral analysis, are available, their use is limited to special cases in which rhythms are regular. To facilitate the analysis of our own episodic LH data, we have developed a process for determining the frequency and amplitude of both regular and irregular endocrine rhythms (signals) in the presence of high levels of random measurement errors (noise). This process, called cycle detection, engages an iterative, computerized procedure which scans data identifying sequential increases and decreases greater than an initial, preset threshold value. One complete cycle is defined as two increases greater than threshold separated by a decrease which is also greater than threshold. For an initial first pass estimate of frequency and amplitude, the threshold is set at 2.7 times the noise standard deviation. On the next pass, the threshold is readjusted, based on an empirically derived formula, and the data are scanned again. This process is repeated until the threshold reaches a stable value. We have tested the reliability of the cycle detection process by simulating irregular rhythm fluctuations of different frequencies, corrupted by various levels of noise and evaluating the signal characteristics with cycle detection analysis. These tests indicate that cycle detection provides excellent estimates of cycle frequency and amplitude, even with signal to noise ratios as low as 1.5. The ability of this process to analyze cyclic signals of almost any shape, with either regular or irregular rhythms, should make it a valuable tool in the hands of endocrine researchers.

Positive and negative feedback effects of ovarian steroids on luteinizing hormone release in ovariectomized rats following chronic depletion of hypothalamic norepinephrine.

The involvement of central noradrenergic mechanisms in the feedback control of LH secretion was studied in ovariectomized rats. Permanent depletion of hypothalamic norepinephrine (NE) was accomplished by transecting the ascending noradrenergic pathway (ANP) in the midbrain. A postcastration elevation in serum LH was observed in rats with complete transections of the ANP, and although the rise was somewhat slower than that seen in sham-cut animals, there was no significant difference between the two groups 5 weeks or more after ovariectomy. Both the positive and negative feedback actions of ovarian steroids remained intact after chronic depletion of NE, and animals with transections of the ANP actually had greater responses than sham-cut controls. In addition, phenoxybenzamine, an alpha-adrenergic receptor blocker which effectively blocked the steroid-induced LH surge in six of eight sham-cut ahimals, was ineffective in NE-depleted rats. These results support the concept that NE acts as a modulator of LH secretion but is not indispensable for feedback control mechanisms.

Pubertal changes in gonadotropin-releasing hormone and proopiomelanocortin gene expression in the brain of the male rat.

Pubertal development in mammals is in part attributable to a brain-dependent process, whereby increased pulsatile GnRH secretion leads to the awakening of the entire reproductive system. However, the brain mechanisms controlling this event are unknown. The apparent increase in GnRH secretion at puberty could reflect an autonomous change in the activity of GnRH neurons themselves or in the afferent networks leading to GnRH neurons. If there were a significant increase in the secretion of GnRH with puberty onset, we hypothesized that there would be a commensurate increase in the biosynthetic capacity of GnRH neurons to meet the increasing demand. We tested this hypothesis by comparing the level of cellular prepro-GnRH mRNA (GnRH mRNA) observed between prepubertal (25-day-old; n = 5) and adult (75-day-old; n = 4) male rats by in situ hybridization. We detected no significant change with puberty in GnRH mRNA signal levels in any of the anatomical areas examined, which included the vertical limb of the diagonal band of Broca, medial septum, lateral preoptic area, and medial preoptic area. Given the variance of our analytical technique, we determined that there was a greater than 90% probability that we would have detected a 20% increase in GnRH mRNA had there been one. Endogenous opioid peptides have been implicated in timing the onset of puberty in the rat, with the argument being that a loss in opioid tone could effect a disinhibition of GnRH secretion. One opioid peptide, beta-endorphin, is among several peptides cleaved from the precursor POMC. We hypothesized that with puberty, POMC neurons in the arcuate nucleus would have an attenuated capacity to produce beta-endorphin. We tested this hypothesis by comparing cellular pre-POMC mRNA (POMC mRNA) levels in the arcuate nuclei of prepubertal (n = 6) and adult (n = 7) male rats with in situ hybridization. We observed an increase in POMC mRNA levels with puberty; prepubertal rats had relative POMC mRNA signal levels of 119 +/- 10 grains/cell, while adult rats contained 167 +/- 12 grains/cell (P less than 0.02). This increase in cellular POMC mRNA was confined to the rostral portion of the arcuate nucleus. We conclude that the GnRH gene is fully expressed well before the time of normal puberty onset and that the increase in POMC mRNA that occurs with the onset of puberty may be important for the development of pulsatile GnRH secretion.

Regulation of luteinizing hormone pulse frequency and amplitude by testosterone in the adult male rat.

Our objective was to gain a better understanding of the role of testosterone (T) in regulating the minute to minute dynamics of LH secretion in the adult male rat. To this end, we examined the patterns of blood LH levels in intact animals and evaluated the effect of small physiological doses of T on mean blood LH and FSH levels and on LH pulse frequency and amplitude in the castrate animal. The intact rat exhibited low frequency (period, approximately 145 min) and low amplitude (approximately 16 ng/ml) LH pulses. After castration, LH pulse frequency (period, approximately 20 min) and amplitude (approximately 118 ng/ml) increased dramatically over that of intact animals. T, administered to castrate rats through Silastic implants, caused a dose-dependent and parallel reduction in mean blood LH and FSH levels. The lowest T dose, which increased mean plasma T levels to 0.5 ng/ml above those of the sham-treated castrates, produced a significant reduction in LH pulse frequency, with a significant increase in pulse amplitude. The next highest T dose caused a reduction in pulse amplitude to a value significantly lower than that in the sham-treated castrates. The highest T dose, which produced steady state mean plasma T levels (approximately 1.6 ng/ml) less than the mean level of the intact group (approximately 2.2 ng/ml), caused a profound reduction in pulse frequency to lower than that of the intact group. These observations demonstrate that T can exert a complex, dose-dependent effect on LH secretory dynamics and imply that one important site of T-mediated negative feedback is the brain's LHRH pulse generator.

Vasoactive intestinal polypeptide effects a central inhibition of pulsatile luteinizing hormone secretion in ovariectomized rats.

Recent studies implicate vasoactive intestinal polypeptide (VIP) as a neurotransmitter or neuromodulator, and several observations suggest that VIP, originating within the brain, may alter the secretion of GnRH. We have tested the hypothesis that VIP acts as a central nervous system regulator of pulsatile GnRH secretion, as reflected in pulsatile LH release, by assessing the effect of intraventricularly administered VIP on several parameters of LH secretion in ovariectomized (OVX) rats. To examine the possibility that centrally administered VIP alters pituitary responsiveness to GnRH, we challenged OVX rats with GnRH and compared the LH response in VIP-treated animals with that in control animals. Finally, we tested the hypothesis that exposure of the animal to gonadal steroids alters VIP's effect on LH secretion by assessing LH release during central administration of VIP in OVX rats pretreated with estrogen and progesterone. Unanesthetized rats with external jugular cannulae were bled at 5-min intervals for 2 h before infusion and for 2 h during continuous intraventricular infusion of either VIP (1.8 nmol/h) or saline. Blood samples (300 or 400 microliter) were replaced with an equal volume of a blood replacement mixture. VIP infusion significantly reduced LH pulse frequency by 80% (P less than 0.002) and mean LH levels by 60% (P less than 0.002), but did not significantly affect LH pulse amplitude. In contrast, saline infusion produced no significant change in any of these parameters. The plasma LH response to 2 ng GnRH, iv, in VIP-treated animals did not differ significantly from that in control animals. Finally, VIP infusion had no discernible effect on LH secretion in OVX rats pretreated with estradiol benzoate (50 micrograms) and progesterone (25 mg). These results demonstrate that VIP can profoundly inhibit pulsatile LH secretion in the OVX rat and provide evidence to suggest that this effect is not due to diminished pituitary responsiveness to GnRH. Based upon these observations, we argue that VIP, originating within the brain, may be an important inhibitory regulator of pulsatile GnRH secretion.

Reduced preprosomatostatin messenger ribonucleic acid in the periventricular nucleus of hypophysectomized rats determined by quantitative in situ hybridization.

Physiological evidence suggests that somatostatin (SS) inhibits the release of GH and TSH from the anterior pituitary and that elements of these two systems may feed back to regulate hypothalamic SS release or synthesis. Hypophysectomy reduces hypothalamic SS content in rats, an effect that may be attributable to a change in SS synthesis, storage, or release. We tested the hypothesis that hypophysectomy would reduce hypothalamic SS synthetic capacity, as reflected by a reduction in SS mRNA levels. Using in situ hybridization and a computerized image analysis system, we measured SS mRNA signal levels over individual cells in the periventricular nucleus of hypophysectomized and intact male rats. SS mRNA signal levels were 45.1% lower in hypophysectomized rats compared to those in intact controls (P less than 0.05). These results demonstrate that SS synthetic capacity in the periventricular nucleus is influenced by the presence of the pituitary, and this system may represent one example of the regulation of central nervous system neuropeptide gene expression by a circulating pituitary hormone.

Sexual dimorphism and testosterone-dependent regulation of somatostatin gene expression in the periventricular nucleus of the rat brain.

Gender differences in hypothalamic somatostatin (SS) secretion may account in part for the sexually dimorphic patterns of GH secretion in rats. Since males have lower baseline serum GH levels than females, and SS inhibits GH secretion, we hypothesized that the SS neurons in the periventricular nucleus (PeN) of the male rat would have greater biosynthetic activity than those of the female. We tested this hypothesis by measuring SS mRNA in cells in the PeN of intact male and proestrous female rats. Using in situ hybridization and a computerized image analysis system, we measured SS mRNA content in individual cells in the PeN and compared signal levels (autoradiographic grains per cell) between male and proestrous female animals. The signal level of SS mRNA in cells of the PeN was significantly greater in males than in proestrous females (males, 210 +/- 7 grains/cell; females, 158 +/- 5 grains/cell; P less than 0.0005), whereas no difference was observed in SS cells of the frontal cortex (males, 100 +/- 0.8 grains/cell; females, 99 +/- 5.9 grains/cell). This difference in SS mRNA levels is likely to be the result of different hormonal environments exerting an influence on neurons of the hypothalamus. To test the hypothesis that testosterone stimulates SS gene expression in neurons of the PeN, adult male rats were castrated and immediately implanted with either empty (sham; n = 3) or testosterone-containing (n = 3) Silastic implants of a size that would deliver physiological levels of testosterone (3.6 +/- 1.5 ng/ml). We observed that castrated animals had significantly lower levels of SS mRNA signal in neurons of the PeN compared with intact animals (intact, 195 +/- 3 grains/cell; castrated, 159 +/- 6 grains/cell; P less than 0.003) and that physiological levels of testosterone prevent this reduction in SS mRNA levels (castrated testosterone-replaced, 182 +/- 4 grains/cell; castrated, 159 +/- 6 grains/cell; P less than 0.003). Furthermore, testosterone-treated castrates had SS mRNA signal levels indistinguishable from those of intact controls (intact, 195 +/- 3 grains/cell; castrated testosterone-replaced, 182 +/- 4 grains/cell). There was no significant difference in SS mRNA levels in neurons of the frontal cortex (intact, 98 +/- 2 grains/cell; castrated, 98 +/- 3 grains/cell; castrated testosterone-replaced, 102 +/- 2 grains/cell).(ABSTRACT TRUNCATED AT 400 WORDS)

Regional specificity of testosterone regulation of proopiomelanocortin gene expression in the arcuate nucleus of the male rat brain.

Endogenous opioid peptides have been implicated as mediators in the negative feedback action of gonadal steroids on GnRH secretion. We have previously demonstrated that testosterone stimulates POMC gene expression in neurons of the arcuate nucleus. However, the wide distribution and variety of actions attributed to the numerous arcuate POMC neurons suggest that these cells may be heterogeneous in their responsiveness to steroid hormones. We tested the hypothesis that testosterone modulates a select population of POMC neurons within the arcuate nucleus of the adult male rat by comparing POMC mRNA signal levels throughout the arcuate nucleus of intact, castrated, and castrated testosterone-replaced adult males. Adult male rats were castrated and implanted (sc) with a Silastic capsule (30 mm) that was either empty (n = 6) or filled with crystalline testosterone (n = 5). Control sham-operated animals (n = 6) were left intact. In each animal the arcuate nucleus was divided into four equal rostral-caudal areas within which we measured POMC mRNA content in individual cells. We report that the effects of castration and testosterone replacement are observed in POMC neurons located in the most rostral region of the arcuate nucleus. After castration, POMC mRNA content was reduced in cells of the most rostral arcuate area (intact, 152 +/- 5 grains/cell; castrate, 119 +/- 2 grains/cell; P less than 0.0005), and replacement with physiological levels of testosterone prevented the decline in POMC mRNA levels so that they remained equivalent to that of the intact animal (castrated testosterone-replaced, 153 +/- 6 grains/cell). There was no significant difference in POMC mRNA signal between intact and castrated testosterone-replaced animals in the most rostral area. POMC neurons in the more caudal aspect of the arcuate (75% of the nucleus) were unaffected by the treatments; alternatively, it is possible that a real change in POMC message content in a subpopulation of cells was obscured by larger numbers of nonresponding cells within the same tissue sections. Based on these observations we conclude that there is a heterogeneous population of POMC neurons in the arcuate nucleus and that testosterone regulates POMC gene expression in a select group of these cells located in the rostral portion of the arcuate nucleus.

The effect of hypophysectomy and growth hormone administration on pre-prosomatostatin messenger ribonucleic acid in the periventricular nucleus of the rat hypothalamus.

Physiological evidence suggests that hypothalamic somatostatin (SS) inhibits pituitary GH release and that GH acts through a short-loop feedback mechanism to stimulate SS secretion. The feedback action of GH could be mediated by an effect on SS synthesis, secretion, or both. We hypothesized that GH acts to regulate the expression of the SS gene and that changes in the level of circulating GH would result in corresponding changes in SS mRNA in cells of the periventricular nucleus (PeN) of the hypothalamus. To test this hypothesis we measured the effect of hypophysectomy (HPX) and HPX with bovine GH (bGH) replacement on SS mRNA signal levels in cells of the PeN of the rat brain. We report that HPX male rats treated with bGH have significantly higher PeN SS mRNA signal than their vehicle-treated controls (P less than 0.05) and that bGH administration to sham-HPX rats results in elevated PeN SS mRNA signal levels compared to those in sham-HPX rats treated with vehicle (P less than 0.05). These observations suggest that GH participates in the regulation of its own secretion by influencing the expression of the SS gene and that one mechanism of short-loop pituitary feedback may involve the modulation of neuropeptide gene expression.

Noradrenergic and endogenous opioid pathways in the regulation of luteinizing hormone secretion in the male rat.

Endogenous opioid pathways (EOPs) appear to play a role in the tonic regulation of pulsatile LH secretion. The mechanism by which EOPs influence LH release is not known. However, the observation that acute chemical disruption of the noradrenergic (NE) system by alpha-receptor-blocking agents or NE synthesis inhibitors blocks the LH increase occurring in response to naloxone (NAL) suggests that the ability of EOPs to influence LH secretion may depend upon a functional NE system. We hypothesized that if EOPs required an interacting NE system to influence LH secretion, then rats that have ascending NE tract lesions would not respond to NAL with an increase in LH. To test this hypothesis, we challenged NE-lesioned and sham-lesioned rats with NAL. Young adult male rats received either a NE lesion or a sham lesion and 6 weeks later were implanted with jugular catheters. On the following day, four baseline blood samples (400 microliters at 10-min intervals) were taken from NE-lesioned and sham-lesioned rats. All rats were then challenged with 2 mg/kg NAL, iv, and four subsequent blood samples were taken. NAL significantly increased LH levels (P less than 0.01) in both the NE-lesioned (n = 7) and sham-lesioned (n = 8) rats. Mean LH responses above baseline for these groups were 0.52 +/- 0.12 ng RP-2/ml and 0.38 +/- 0.07 ng/ml, respectively. Failure of the NE lesions to block the LH response to NAL was not due to the presence of active residual NE fibers, because pretreatment with the alpha-receptor blocker phenoxybenzamine (20 mg/kg, iv) failed to block the NAL response in the NE-lesioned animals (n = 8), whereas it did so in the sham-lesioned animals (n = 7). The mean LH response from baseline after phenoxybenzamine pretreatment was -0.01 +/- 0.01 ng/ml for the sham-lesioned group and 0.53 +/- 0.24 ng/ml for the NE-lesioned group. These results indicate that EOPs do not require noradrenergic mediation to influence LH secretion in the adult male rat.

Gonadotropin-releasing hormone messenger ribonucleic acid levels are unaltered with changes in the gonadal hormone milieu of the adult male rat.

Testicular function is regulated by the negative feedback effect of sex hormones acting at the brain and pituitary to inhibit the secretion of LH and FSH. An important component of this feedback axis is presumed to involve regulation of secretion and possibly synthesis of GnRH by the brain. We tested the hypothesis that the castration-induced increase in gonadotropin secretion is subserved, at least in part, by increased synthesis of GnRH. Using in situ hybridization and an oligonucleotide probe to pro-GnRH messenger RNA (GnRH mRNA), we compared the level of cellular GnRH mRNA and the relative number of GnRH mRNA-containing neurons between intact and 21-day castrate adult male rats. To derive estimates of the number of GnRH cells and the cellular GnRH mRNA content, coronal sections from each animal were anatomically matched between intact and castrate groups. All identifiable cells within these sections were counted and analyzed with the aid of a computerized image analysis system, by an observer unaware of the animal's experimental group and were assigned an anatomical location for reference. In an initial experiment, we observed no difference in cellular GnRH mRNA signal level between intact (n = 4) and castrate (n = 5) animals (129 +/- 8 vs. 139 +/- 5 grains per cell); however, we did find a statistical difference between the intact and castrated groups in the relative number of GnRH mRNA-containing cells (intact: 212 +/- 15 vs. castrate: 320 +/- 18). To confirm this observation, we repeated the experiment by again comparing the number of GnRH mRNA-positive cells between intact (n = 4) and castrate (n = 4) rats. In this second experiment, we found no difference in the number of identifiable GnRH mRNA-containing cells between intact and castrate animals (272 +/- 14 vs. 274 +/- 36, respectively); this was the case for the total cell count as well as when the data were analyzed by anatomical region. To clarify the conflicting results on cell counts of Exps 1 and 2, we repeated the experiment a third time, again comparing both the number of GnRH mRNA-containing cells and the cellular content of GnRH mRNA. In this experiment, we observed that neither cell number nor content of GnRH mRNA differed between the intact and castrate groups. Again, this was the case for total cell count, as well as when the data were analyzed by anatomical region.(ABSTRACT TRUNCATED AT 400 WORDS)

Testosterone regulates pro-opiomelanocortin gene expression in the primate brain.

Endogenous opioid peptides such as beta-endorphin, derived from proopiomelanocortin (POMC), have been widely implicated as serving an important role in the neuroendocrine regulation of the primate reproductive axis. In both human and nonhuman primates, POMC neurons are thought to mediate, at least in part, the negative feedback action of sex steroids on GnRH. Sex steroids, such as testosterone, are thought to inhibit GnRH secretion by enhancing the inhibitory activity of beta-endorphin; however, the cellular mechanisms by which steroid hormones regulate the activity of POMC neurons in the primate brain are unknown. In this study, we tested the hypothesis that testosterone stimulates POMC gene expression within the primate brain and that this regulation occurs within a specific subset of POMC neurons residing in the arcuate nucleus of the hypothalamus. We used in situ hybridization to compare cellular levels of POMC messenger RNA in intact (n = 4), castrated (n = 4), and castrated/testosterone-treated (n = 4) monkeys. We report that after castration of the male macaque (Macaca fascicularis), cellular POMC messenger RNA levels decline significantly (P less than 0.05) in neurons within the arcuate nucleus and that this decline is prevented by replacement with physiological doses of testosterone. Moreover, we found that this testosterone-dependent modulation of POMC gene expression is restricted to a small fraction of the numerous POMC neurons located within the most anterior region of the arcuate nucleus in the brain of this primate species. These observations provide evidence that sex steroids regulate expression of the POMC gene in the primate brain.

Regulation of hypothalamic neuropeptide-Y neurons by growth hormone in the rat.

GH is thought to exert a short-loop feedback action on the hypothalamic somatostatin- and GH-releasing hormone (GHRH)-containing neurons. The direct actions of GH are mediated through GH receptors. In the male rat, few GHRH-containing neurons in the arcuate nucleus (ARC) appear to express the GH receptor messenger RNA (mRNA); however, some unidentified neurons near GHRH neurons do. Recent evidence suggests that neuropeptide-Y (NPY)-containing neurons, which are located near GHRH neurons in the ARC, are targets for GH action because treatment of rats with GH induces c-fos expression in these cells. We conducted two experiments to test the hypothesis that GH acts on NPY neurons in the ARC. First, we performed double-label in situ hybridization to determine whether NPY neurons in the ARC express GH receptor mRNA. Second, we investigated the possibility that GH regulates NPY mRNA expression by using in situ hybridization to compare ARC NPY mRNA levels among groups of normal (n = 7), hypophysectomized (n = 7), and hypophysectomized/rGH-treated (1.5 mg rat GH over 3 days; n = 6) rats. We found that most of the NPY-containing neurons in the ARC expressed GH receptor mRNA, whereas hypothalamic NPY neurons residing outside of the ARC did not. Furthermore, hypophysectomy significantly decreased NPY mRNA levels, and GH treatment restored the levels to those of the intact animals. We conclude that GH regulates the activity of NPY neurons in the ARC by a direct action on GH receptors that are expressed by NPY neurons. Whether the action of GH on NPY neurons in the ARC is related to the feedback control of GH secretion or some other physiological function remains to be determined.

Proopiomelanocortin neurons are direct targets for leptin in the hypothalamus.

Leptin is a protein product of the obese (ob) gene, which is secreted by adipocytes and functions as a satiety factor to regulate food intake. The expression of the leptin receptor in several hypothalamic nuclei suggests that multiple neuronal subtypes are targets for leptin's action. Products of the proopiomelanocortin (POMC) gene are known to affect feeding behavior, and POMC neurons share a similar distribution with leptin receptor mRNA in the arcuate nucleus. We used double label in situ hybridization and computerized image analysis to test the hypothesis that POMC neurons coexpress the leptin receptor. Quantitative analysis confirmed that POMC neurons in the hypothalamus express leptin receptor mRNA. Based on this observation, we infer that POMC neurons and the products of the POMC gene may be part of the signaling pathway mediating leptin's action on feeding and perhaps other physiological functions.

Sexual dimorphism of growth hormone-releasing hormone and somatostatin gene expression in the hypothalamus of the rat during development.

The secretory pattern of GH secretion is markedly sexually dimorphic in the adult rat. The patterning of GH secretion is determined by the coordinated activity of somatostatin (SS)- and GH-releasing hormone (GHRH)-containing neurosecretory cells located in the hypothalamus. In this study we examined whether there is sexual dimorphism in the expression of the SS and GHRH genes and, if so, at what developmental stage this becomes evident. To address these questions, we measured SS messenger RNA (mRNA) levels in neurons of the periventricular nucleus and GHRH mRNA levels in the arcuate nucleus and ventromedial nucleus of the hypothalamus in male and female rats at 10, 25, 35, and 75 days of age. Using in situ hybridization and a computerized image analysis system, we measured SS mRNA and GHRH mRNA signal levels in individual neurons and compared these levels among the different age groups. We found that male animals had significantly higher levels of SS mRNA than females at every age. Similarly, males had higher GHRH mRNA levels than females; however, this difference was statistically significant only at 10 and 75 days of age. Developmental changes in GHRH mRNA levels were similar for both sexes, with GHRH message levels increasing gradually over the course of maturation. SS mRNA signal levels also changed over the course of development in both male and female animals. In the male rat, SS mRNA levels increased significantly between 10 and 25 days of age and declined significantly between 35 and 75 days of age. In the female rat, SS mRNA levels increased gradually between 10 and 35 days of age, then, as in the male, declined significantly between days 35 and 75. We conclude that sex differences and age-dependent changes in the expression of the SS and GHRH genes may subserve the sexual dimorphism and developmental alterations in the pattern of GH secretion in the rat.