Parathyroid hormone messenger ribonucleic acid in the rat hypothalamus

Polyadenylated RNA, extracted from rat hypothalami, cross-hybridized with a RNA probe complementary in sequence to rat PTH (rPTH) messenger RNA (mRNA). Amplification of complementary DNA (cDNA) by the polymerase chain reaction also demonstrated the presence of rPTH mRNA in the rat hypothalamus and parathyroid gland. rPTH mRNA was localized by in situ hybridization in the paraventricular and supraoptic nuclei of the rat hypothalamus. These results demonstrate the expression of the PTH gene in the central nervous system of the rat in areas which suggest roles for PTH in neuroendocrine function.     

Growth hormone receptor messenger ribonucleic acid distribution in the adult male rat brain and its colocalization in hypothalamic somatostatin neurons.

The activity of both somatostatin (SS) and GH-releasing hormone (GHRH) neurons within several hypothalamic nuclei is regulated, in part, by the feedback effects of GH. However, whether GH, or its intermediate, insulin-like growth factor I, acts on these neurons to alter the synthesis and release of SS and GHRH is unknown. We argued that if GH itself acts directly on the brain to govern its own secretion, then regions of the brain containing SS and GHRH neurons may express the GH receptor gene. We tested this hypothesis by performing in situ hybridization for GH receptor messenger RNA (mRNA) and mapping its distribution in the brain. We observed GH receptor mRNA-containing cells in various brain regions including the thalamus, septal region, hippocampus, dentate gyrus, amygdala, and hypothalamus. Next we sought evidence for expression of the GH receptor mRNA by SS neurons in the hypothalamus. We addressed this by performing a double-label in situ hybridization to identify neurons expressing both SS mRNA and GH receptor mRNA. We report that SS neurons in the periventricular nucleus and in the paraventricular nucleus coexpress the GH receptor gene, whereas few, if any, of the SS neurons in the cortex express detectable amounts of the GH receptor mRNA. These findings suggest that GH acts directly on the brain and participates in the regulation of its own secretion through a direct action on hypothalamic SS neurons.     

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)     

The rat melanin-concentrating hormone messenger ribonucleic acid encodes multiple putative neuropeptides coexpressed in the dorsolateral hypothalamus

The melanin-concentrating hormone (MCH) is a cyclic neuropeptide, first isolated from salmon pituitary glands, which regulates melanin dispersion in the skin and perhaps the activity of the pituitary-adrenal axis in teleost fish. We have recently purified and characterized rat MCH (rMCH) and report here the cloning and sequencing of specific MCH cDNA isolated from a rat hypothalamic library. The sequence of rMCH found by DNA sequencing confirms the sequence deduced from the purified peptide. rMCH is located at the C-terminus of a protein precursor of 165 amino acid residues. Comparison of the amino acid sequence of prepro-MCH and that of the Aplysia peptide-A prohormone suggests that these proteins as well as other precursors may be evolutionarily related. Besides rMCH, two putative neuropeptides, termed NGE and NEI, might be generated from the same precursor. The rMCH precursor shared sequence identities with human GH-releasing factor and mammalian CRF in the regions encoding NGE and NEI. By immunohistochemical studies we have established that the amidated C-terminus of NEI is recognized by some alpha MSH and rat CRF antisera and that the C-terminal portion of NGE is responsible for the cross-reactivity revealed with one hGRF-(1-37) antiserum. Our results explain the staining of a discrete population of dorso-lateral hypothalamic neurons by heretofore seemingly unrelated antisera and provide evidence for the production of multiple novel neuropeptides from a common precursor.     

Regulation of rat hypothalamic preprogrowth hormone-releasing factor messenger ribonucleic acid by dietary protein

To further evaluate nutrient regulation of GRF synthesis, we measured hypothalamic preproGRF messenger (m) RNA in food-deprived rats refed diets varying in nutrient composition by nuclease protection analysis. Adult male Sprague-Dawley rats were allowed free access to food (Fed), food deprived for 72 h (72-h FD), or 72 h FD then refed for 72 h with either a normal (NF) diet or isocaloric diets containing no protein (PF), carbohydrate (CF), or fat (FF). Seventy-two-hour FD rats displayed the expected 80% reduction in hypothalamic preproGRF mRNA. Upon refeeding, levels were normalized in rats refed NF, CF, or FF diets. In contrast, preproGRF mRNA in rats refed a PF diet was similar to that in 72-h FD rats. Rats refed a PF diet failed to gain weight and consumed less food than animals refed NF, CF, or FF diets. However, the lack of the GRF response to the PF diet was due to protein deprivation rather than caloric restriction, since hypothalamic preproGRF mRNA returned to 66% of Fed values in rats refed an equivalent amount (grams per day) of a NF diet. In 72-h FD rats refed isocaloric diets containing 4%, 8%, or 12% protein, preproGRF mRNA was restored to Fed values in a protein concentration-dependent manner being completely restored by the 12% diet. A lack of dietary protein was sufficient to regulate hypothalamic preproGRF mRNA since feeding rats a PF diet without prior food deprivation resulted in 70% reduction in preproGRF mRNA, whereas CF and FF diets were without effect. These data indicate that decreased hypothalamic preproGRF mRNA expression in 72-h FD rats occurs as a result of dietary protein deprivation.     

Insulin-induced hypoglycemia increases corticotropin-releasing factor messenger ribonucleic acid levels in rat hypothalamus

To study the effect of acute stress on CRF release and synthesis in rat hypothalamus, ACTH levels in plasma, CRF contents in the median eminence (ME), and CRF mRNA levels in the hypothalamus without ME and cerebral cortex were determined after insulin-induced hypoglycemia. Plasma ACTH levels increased at 30 and 60 min, while ME CRF content decreased at 30 and 60 min, then returned to the control level at 90 min. Hybridization with a cRNA probe revealed a single size class of CRF mRNA in the hypothalamus and cerebral cortex (approximately 1300 nucleotides), and the size of CRF mRNA in these tissues did not change during the experimental period. CRF mRNA levels in the hypothalamus increased to 130% of the control value at 30 min and reached a peak (186% of the control value) at 120 min, but these levels in the cerebral cortex did not change. These results suggest that insulin-induced hypoglycemia stimulates CRF synthesis by increasing CRF mRNA levels in the hypothalamus as well as CRF release, and that release and synthesis of CRF in the cerebral cortex are independent of those in the hypothalamus.     

Hypothalamic prolactin stimulates the release of luteinizing hormone-releasing hormone from male rat hypothalamus

Previous works from our laboratory and others have shown that there is a PRL-like immunoreactive protein with immunological, chromatographic, and biological characteristics identical to those of pituitary PRL, and this is widely distributed in the rat central nervous system. Since pituitary PRL is important in controlling hypothalamic LHRH release, we have hypothesized that hypothalamic PRL-like immunoreactive protein might serve a similar role, that of an endogenous neuromodulator influencing hypothalamic LHRH release. To this end, we have examined the effect of PRL antiserum and normal rabbit serum on the release of immunoreactive LHRH from rat hypothalamic fragments cultured in vitro. In the first experiment, LHRH release from hypothalami of intact rats, bathed in PRL antiserum (1:200 in Krebs-Ringer bicarbonate buffer), was significantly lower than that from hypothalami bathed in normal rabbit serum (1:200 in Krebs-Ringer bicarbonate buffer) for 90 min of incubation. It was, however, possible that the PRL, immunoneutralized in the first experiment, was material that represented contamination from pituitary PRL. Therefore, we repeated the experiment using hypothalami from animals that had been hypophysectomized 2 weeks before death. Again, PRL antibody significantly inhibited the release of LHRH compared with that by hypothalami incubated in normal rabbit serum. Since testosterone is important to LHRH synthesis, a third experiment was carried out using hypothalami from hypophysectomized male rats that had been implanted sc with testosterone-containing capsules 72 h before death. By 72 h serum testosterone levels had normalized. PRL antibody added to medium containing hypothalamic explants from these animals substantially inhibited in vitro LHRH release, a pattern essentially similar to that seen in intact and hypophysectomized animals without testosterone replacement. From these studies we have concluded that hypothalamic PRL is an important neuromodulator that promotes the release of LHRH from the hypothalamus. Testosterone, at least under the experimental conditions employed, appears not to be essential in this hypothalamic PRL-LHRH interaction.     

Glucocorticoids decrease thyrotropin-releasing hormone messenger ribonucleic acid expression in the paraventricular nucleus of the human hypothalamus

The way glucocorticoids affect TRH mRNA expression in the paraventricular nucleus of the hypothalamus is still unclear. In view of its relevance for Cushing's syndrome and depression, we measured TRH mRNA expression in human hypothalami obtained at autopsy by means of quantitative TRH mRNA in situ hybridization. In corticosteroid-treated subjects (n = 10), TRH mRNA hybridization signal was decreased as compared with matched control subjects (n = 10) (Mann-Whitney U test, P = 0.02). By inference, hypercortisolism as present in patients with Cushing's syndrome or major depression may contribute to lower serum TSH or symptoms of depression by lowering hypothalamic TRH expression.     

Single cell levels of hypothalamic messenger ribonucleic acid encoding luteinizing hormone-releasing hormone in intact, castrated, and hyperprolactinemic male rats

We have examined the changes that occur in neuronal expression of LHRH mRNA in response to castration and hyperprolactinemia in male rats. Single cell levels of LHRH mRNA were determined by quantitative in situ hybridization histochemistry using an 35S-labeled synthetic 48-base oligodeoxynucleotide probe and quantitative autoradiography. Nine days postcastration, a 10.4-fold increase in mean plasma LH titers was observed which was associated with significantly increased LHRH mRNA in rostral hypothalamic neuronal cell bodies. Both increases were blocked in rats rendered hyperprolactinemic by the presence of the 7315a PRL-secreting pituitary tumor. The location and number of neurons expressing LHRH mRNA were unchanged, indicating that these differences were attributable to amounts of mRNA expressed per neuron. Experimental differences occurred in LHRH perikarya situated throughout the rostral hypothalamus from the organum vasculosum of the lamina terminalis to the caudal extent of the medial preoptic nucleus. These results suggest that gonadal steroids and PRL are involved, either directly or indirectly, in regulating the biosynthesis of LHRH in the rostral hypothalamus.     

Detection of estrogen receptor-beta messenger ribonucleic acid and 125I-estrogen binding sites in luteinizing hormone-releasing hormone neurons of the rat brain

Luteinizing hormone-releasing hormone (LHRH) neurons of the forebrain play a pivotal role in the neuroendocrine control of reproduction. Although serum estrogen levels influence many aspects of LHRH neuronal activity in the female, earlier studies were unable to detect estrogen receptors (ERs) within LHRH neurons, thus shaping a consensus view that the effects of estradiol on the LHRH neuronal system are mediated by interneurons and/or the glial matrix. The present studies used dual-label in situ hybridization histochemistry (ISHH) and combined LHRH-immunocytochemistry/125I-estrogen binding to readdress the estrogen-receptivity of LHRH neurons in the female rat. In ISHH experiments we found that the majority of LHRH neurons exhibited hybridization signal for the "beta" form of ER (ER-beta). The degree of colocalization was similar in topographically distinct populations of LHRH neurons and was not significantly altered by estradiol (67.2+/-1.8% in ovariectomized and 73.8+/-4.2% in ovariectomized and estradiol-treated rats). In contrast, the mRNA encoding the classical ER-alpha could not be detected within LHRH neurons. In addition, in vivo binding studies using 125I-estrogen revealed a subset of LHRH-immunoreactive neurons (8.8%) which accumulated the radioligand thus providing evidence for the translation of ER protein(s) within these cells. The findings that most LHRH neurons in the female rat express ER-beta mRNA and at least some are capable of binding 125I-estrogen challenge the current opinion that estrogen does not exert direct effects upon the LHRH neuronal system.     

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.     

Angiotensin II increases the corticotropin-releasing factor messenger ribonucleic acid level in the rat hypothalamus.

Angiotensin II (AII) has an important role in the regulation of CRF release. In the present study, the effect of centrally administered AII on CRF messenger RNA (mRNA) levels in the rat hypothalamus was examined. Administration of 0.1 nmol and 1 nmol AII into the lateral ventricle increased the levels of plasma ACTH 20 min and 45 min after administration and those of proopiomelanocortin mRNA in the anterior pituitary (AP) and CRF mRNA in the hypothalamus 2 h after administration. On the other hand, ACTH levels in AP and CRF levels in the median eminence temporarily decreased 45 min after the administration of 1 nmol AII, but it returned to the control level at 90 min. Administration of 10 nmol saralacin, an AII antagonist, blocked 1 nmol AII-induced increase in the levels of plasma ACTH, proopiomelanocortin mRNA in AP, and CRF mRNA in the hypothalamus. These results indicate that central administration of AII increases the CRF mRNA level in the hypothalamus in a receptor-specific manner and also increases CRF release. Therefore, AII seems to have an important role in the regulation of the release and synthesis of CRF in the hypothalamus.     

Hypotensive hemorrhage elevates corticotropin-releasing hormone messenger ribonucleic acid (mRNA) but not vasopressin mRNA in the rat hypothalamus.

We examined the effect of acute hypotensive hemorrhage on corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) messenger RNAs (mRNAs) in neurons of the rat hypothalamus. Sprague-Dawley male rats were cannulated (femoral artery and vein) and received a 15 ml/kg.3 min hemorrhage on the morning of the fourth day. Time controls received no hemorrhage. After light halothane anesthesia, the rats were decapitated at 1 or 4 h (six to nine rats per group). The hypothalami were removed, frozen, and sectioned at 12 microns. In situ hybridization was performed using two 48-base oligodeoxynucleotide probes for CRH and AVP message, respectively. Hemorrhage led to a fall in arterial blood pressure and heart rate that recovered by 1 h. Plasma ACTH, corticosterone, and AVP were elevated 20, 60, and 90 min after hemorrhage, but returned to near control levels by 4 h. CRH mRNA was significantly elevated 1 and 4 h after hemorrhage, as compared to time controls, in parvocellular neurons of the paraventricular nuclei. However, AVP mRNA was not different from controls at 1 or 4 h after hemorrhage in the magnocellular or parvocellular paraventricular nuclei, or in the supraoptic or accessory nuclei of the hypothalamus. AVP mRNA was also found in neurons of the suprachiasmatic nuclei, but there was no difference in the amount of mRNA between the 1-h hemorrhage and control groups. These data suggest that neural signals, originating for cardiovascular receptors activated by hemorrhage, up-regulate message for CRH but not for AVP in the paraventricular nuclei of the rat hypothalamus.     

Activation of SOCS-3 messenger ribonucleic acid in the hypothalamus by ciliary neurotrophic factor

Ciliary neurotrophic factor (CNTF) is a neurocytokine expressed in glial cells that acts on brain cells to promote gene expression, survival, and differentiation. When administered systemically, CNTF reduces food intake and body weight in rodents. Genes encoding suppressors of cytokine signaling (SOCS) are induced by cytokines that activate membrane receptors in the same class as those that are activated by CNTF. We therefore examined the ability of CNTF to induce expression of socs genes in brain and peripheral tissues of rats and mice. Peripheral CNTF administration to ob/ob mice rapidly induced SOCS-3 messenger RNA (mRNA) in hypothalamus, as determined by Northern blotting and quantitative RT-PCR, but had no effect on cytokine-inducible sequence (CIS), SOCS-1, or SOCS-2 mRNA. In situ hybridization histochemistry of hypothalamus from ob/ob mice and normal rats demonstrated that CNTF induced SOCS-3 mRNA in the arcuate nucleus (Arc). Strong hybridization signals were also detected in the ependymal lining of the ventricles and the subfornical organ. This hybridization pattern was distinct from that resulting from peripheral leptin treatment with overlapping hybridization patterns only in the Arc. CNTF also induced expression of CIS, SOCS-1, SOCS-2, and SOCS-3 mRNA in the liver, and SOCS-2 and SOCS-3 mRNA in the kidney. CNTF induced SOCS-3 mRNA and SOCS-3 protein levels in an astrocyte cell line. Transient expression of SOCS-3, but not CIS or SOCS-2, inhibited CNTF-induced signal transduction in astrocytes. In conclusion, SOCS-3 mRNA is specifically induced by CNTF in regions of the hypothalamus that are both overlapping and distinct from that induced by leptin. Similar to leptin, the Arc is likely to be a direct target of CNTF, and this region may play a role in the body weight-reducing effects of CNTF. SOCS-3 is a negative regulator of CNTF signal transduction, and inhibitors of SOCS-3 function may enhance endogenous CNTF signaling after neuronal injury or enhance the body weight-reducing effect of CNTF after peripheral administration.     

Ontogenesis of growth hormone-releasing hormone neurons in the rat hypothalamus

The ontogenesis of growth hormone releasing hormone (GH-RH) containing neurons in the rat hypothalamus has been studied by immunohistochemistry, using a specific anti-rat GH-RH serum. Immunoreactive fibers were first detected in the prospective median eminence on day 18 of gestation. During the subsequent 3 days, they rapidly increased in distribution and intensity of staining within this structure. On day 21, positive fibers were also visible in a plexus within the arcuate nucleus. In 1-day-old rats treated with colchicine, positive perikarya were distributed in several hypothalamic nuclei, including the arcuate nucleus, dorsomedial nucleus, basal lateral hypothalamus, and perifornical region. The distribution was similar to that previously described in adult rats. The intensity of staining in the various hypothalamic regions increased during early postnatal life to levels nearly comparable to those in adult rats by 30 days. These findings showing the early appearance of GH-RH-positive terminals in the median eminence and the wide distribution of the perikarya at an early stage of postnatal life support the view that hypothalamic GH-RH serves an important role in the regulation of growth hormone secretion during late prenatal and early neonatal periods.     

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.     

Testosterone regulation of proopiomelanocortin messenger ribonucleic acid in the arcuate nucleus of the male rat

GnRH regulates the secretion of LH and FSH, which stimulate the secretion of testicular hormones. Acting in a reciprocal fashion, these hormones, including testosterone and inhibin, exert a negative feedback effect on GnRH and gonadotropin secretion. Endogenous opioid peptides (EOPs) have been implicated to play a role in steroid-mediated regulation of gonadotropin secretion. In this context, certain steroid hormones (e.g. testosterone) increase EOP activity and ultimately inhibit GnRH secretion; however, the cellular mechanism by which this occurs is unknown. beta-Endorphin is one of these EOPs, and it is derived from a larger precursor molecule, POMC. We tested the hypothesis that testicular hormones and testosterone, in particular, stimulate POMC gene expression in the arcuate nucleus of the male rat brain. First, we compared POMC mRNA levels between intact and castrated male rats. Adult male rats were killed 4 days (n = 4) and 21 days (n = 5) after castration. Intact animals (sham-operated; n = 6) were used as controls. Using in situ hybridization and a computerized image analysis system, we measured the POMC mRNA content in individual cells of the arcuate nucleus. POMC mRNA signal was significantly lower (P less than 0.0003) in both 4-day (126 +/- 2 grains/cell) and 21-day (117 +/- 5 grains/cell) castrates than in controls (142 +/- 2 grains/cell). In a second experiment we tested whether testosterone would reverse the castration-induced loss of POMC message. Again, we castrated animals and immediately implanted them with either empty (sham; n = 6) or testosterone-containing Silastic implants (n = 5) of a size that would deliver physiological levels of testosterone (3.6 +/- 1.5 ng/ml). We observed that testosterone-treated animals had significantly higher levels of POMC mRNA signal (121.8 +/- 3.8 grains/cell) than sham-treated castrates (111.4 +/- 3.6 grains/cell; P less than 0.03) and that the testosterone-treated castrates had POMC mRNA signal levels indistinguishable from those of intact controls (122.0 +/- 1.1 grains/cell). These observations lend credence to the theory that one mechanism by which testosterone may regulate GnRH secretion is by increasing the synthesis of POMC in the arcuate nucleus.     

KiSS-1 messenger ribonucleic acid expression in the hypothalamus of the ewe is regulated by sex steroids and season

The KiSS-1 gene encodes a family of peptides called kisspeptins, which are endogenous ligands for the G protein-coupled receptor GPR54. Kisspeptin function appears to be critical for GnRH secretion and the initiation of puberty. To test the hypothesis that steroid hormones regulate KiSS-1 mRNA expression in the ewe, we examined the brains of ovary-intact (luteal phase) and ovariectomized (OVX) ewes, as well as OVX ewes that received estradiol (E) or progesterone (P) replacement. KiSS-1 mRNA-expressing cells were predominantly located in the arcuate nucleus (ARC). Here, expression was increased after OVX but returned to the level of gonad-intact animals with E treatment. Treatment with P partially restored KiSS-1 expression toward gonad-intact levels. Double-label immunohistochemistry revealed that approximately 86% of kisspeptin-immunoreactive cells in the ARC are also P-receptor positive. Finally, we tested the hypothesis that KiSS-1 mRNA is lower during anestrus, due to a non-steroid-dependent seasonal effect. In OVX ewes, expression in the ARC was lower at the time of year corresponding to anestrus. We conclude that KiSS-1 expression in the ARC of the ewe brain is negatively regulated by chronic levels of E and P, suggesting that both steroids may exert negative feedback control on GnRH secretion through altered kisspeptin signaling. Furthermore, a seasonal alteration in KiSS-1 expression in the ARC of OVX ewes strongly suggests that kisspeptin is fundamentally involved in the control of seasonal changes in reproductive function.