The Emerging Therapeutic Potential of Kisspeptin and Neurokinin B.

Kisspeptin (KP) and neurokinin B (NKB) are neuropeptides that govern the reproductive endocrine axis through regulating hypothalamic gonadotropin-releasing hormone (GnRH) neuronal activity and pulsatile GnRH secretion. Their critical role in reproductive health was first identified after inactivating variants in genes encoding for KP or NKB signaling were shown to result in congenital hypogonadotropic hypogonadism and a failure of pubertal development. Over the past 2 decades since their discovery, a wealth of evidence from both basic and translational research has laid the foundation for potential therapeutic applications. Beyond KP's function in the hypothalamus, it is also expressed in the placenta, liver, pancreas, adipose tissue, bone, and limbic regions, giving rise to several avenues of research for use in the diagnosis and treatment of pregnancy, metabolic, liver, bone, and behavioral disorders. The role played by NKB in stimulating the hypothalamic thermoregulatory center to mediate menopausal hot flashes has led to the development of medications that antagonize its action as a novel nonsteroidal therapeutic agent for this indication. Furthermore, the ability of NKB antagonism to partially suppress (but not abolish) the reproductive endocrine axis has supported its potential use for the treatment of various reproductive disorders including polycystic ovary syndrome, uterine fibroids, and endometriosis. This review will provide a comprehensive up-to-date overview of the preclinical and clinical data that have paved the way for the development of diagnostic and therapeutic applications of KP and NKB.

Hypothalamic Overexpression of Makorin Ring Finger Protein 3 Results in Delayed Puberty in Female Mice.

Makorin ring finger protein 3 (MKRN3) is an important neuroendocrine player in the control of pubertal timing and upstream inhibitor of gonadotropin-releasing hormone secretion. In mice, expression of Mkrn3 in the hypothalamic arcuate and anteroventral periventricular nucleus is high early in life and declines before the onset of puberty. Therefore, we aimed to explore if the persistence of hypothalamic Mkrn3 expression peripubertally would result in delayed puberty. Female mice that received neonatal bilateral intracerebroventricular injections of a recombinant adeno-associated virus expressing Mkrn3 had delayed vaginal opening and first estrus compared with animals injected with control virus. Subsequent estrous cycles and fertility were normal. Interestingly, male mice treated similarly did not exhibit delayed puberty onset. Kiss1, Tac2, and Pdyn mRNA levels were increased in the mediobasal hypothalamus in females at postnatal day 28, whereas kisspeptin and neurokinin B protein levels in the arcuate nucleus were decreased, following Mkrn3 overexpression, compared to controls. Cumulatively, these data suggest that Mkrn3 may directly or indirectly target neuropeptides of Kiss1 neurons to degradation pathways. This mouse model suggests that MKRN3 may be a potential contributor to delayed onset of puberty, in addition to its well-established roles in central precocious puberty and the timing of menarche.

Hyperandrogenism induces proportional changes in the expression of Kiss-1, Tac2, and DynA in hypothalamic KNDy neurons.

BACKGROUND: Kisspeptin released from Kiss-1 neurons in the hypothalamus plays an essential role in the control of the hypothalamic-pituitary-gonadal axis by regulating the release of gonadotropin-releasing hormone (GnRH). In this study, we examined how androgen supplementation affects the characteristics of Kiss-1 neurons. METHODS: We used a Kiss-1-expressing mHypoA-55 cell model that originated from the arcuate nucleus (ARC) of the mouse hypothalamus. These cells are KNDy neurons that co-express neurokinin B (NKB) and dynorphin A (DynA). We stimulated these cells with androgens and examined them. We also examined the ARC region of the hypothalamus in ovary-intact female rats after supplementation with androgens. RESULTS: Stimulation of mHypoA-55 cells with 100 nM testosterone significantly increased Kiss-1 gene expression by 3.20 ± 0.44-fold; testosterone also increased kisspeptin protein expression. The expression of Tac3, the gene encoding NKB, was also increased by 2.69 ± 0.64-fold following stimulation of mHypoA-55 cells with 100 nM testosterone. DynA gene expression in these cells was unchanged by testosterone stimulation, but it was significantly reduced at the protein level. Dihydrotestosterone (DHT) had a similar effect to testosterone in mHypoA-55 cells; kisspeptin and NKB protein expression was significantly increased by DHT, whereas it significantly reduced DynA expression. In ovary-intact female rats, DTH administration significantly increased the gene expression of Kiss-1 and Tac3, but not DynA, in the arcuate nucleus. Exogenous NKB and DynA stimulation failed to modulate Kiss-1 gene expression in mHypoA-55 cells. Unlike androgen stimulation, prolactin stimulation did not modulate kisspeptin, NKB, or DynA protein expression in these cells. CONCLUSIONS: Our observations imply that hyperandrogenemia affects KNDy neurons and changes their neuronal characteristics by increasing kisspeptin and NKB levels and decreasing DynA levels. These changes might cause dysfunction of the hypothalamic-pituitary-gonadal axis.

Green tea catechin EGCG could prevent obesity-related precocious puberty through NKB/NK3R signaling pathway.

This study aimed to explore the potential regulatory pathways of (-)-epigallocatechin-3-gallate (EGCG) in preventing obesity-related precocious puberty. A retrospective analysis on the impact of EGCG on puberty onset in obese girls was conducted on plasma samples collected from a human randomized controlled trial. In the trial, participants consumed EGCG capsules for 12 weeks. In the animal experiment, rats were divided into four groups: normal diet control (NC) group, high-fat diet (HFD) group, NC+EGCG group, and HFD+EGCG group. Blood samples were collected on postnatal days 27, 33, and 36 to detect sexual development indicators. The hypothalamic expressions of kisspeptin/Kiss1R and neurokinin B (NKB)/NK3R signaling were measured by RT-qPCR and Western blot assay. The ovary NKB protein expression was assessed by immunohistochemical assays. Serum NKB level in the EGCG group was lower than the placebo group by 0.599 ng/mL [ß=-0.599, 95% CI: (-1.005, -0.193)], at the end of intervention and after adjusting for confounders (clinical study). In the animal experiment, EGCG intervention could significantly delay the vaginal opening (VO) time of rats fed with HFD. On day 33, EGCG intervention could significantly reduce serum NKB, luteinizing hormone (LH) levels, ovarian NKB protein expression, and endometrial thickness of HFD-fed rats, while EGCG intervention could remarkably increase mRNA and protein expression of NKB/NK3R. EGCG could prevent obesity-related precocious puberty through NKB/NK3R signaling pathway, which may provide a novel insight into the role of EGCG in preventing precocious puberty in obese girls.

Localization of kisspeptin, NKB, and NK3R in the hypothalamus of gilts treated with the progestin altrenogest.

Mechanisms in the brain controlling secretion of gonadotropin hormones in pigs, particularly luteinizing hormone (LH), are poorly understood. Kisspeptin is a potent LH stimulant that is essential for fertility in many species, including pigs. Neurokinin B (NKB) acting through neurokinin 3 receptor (NK3R) is involved in kisspeptin-stimulated LH release, but organization of NKB and NK3R within the porcine hypothalamus is unknown. Hypothalamic tissue from ovariectomized (OVX) gilts was used to determine the distribution of immunoreactive kisspeptin, NKB, and NK3R cells in the arcuate nucleus (ARC). Almost all kisspeptin neurons coexpressed NKB in the porcine ARC. Immunostaining for NK3R was distributed throughout the preoptic area (POA) and in several hypothalamic areas including the periventricular and retrochiasmatic areas but was not detected within the ARC. There was no colocalization of NK3R with gonadotropin-releasing hormone (GnRH), but NK3R-positive fibers in the POA were in close apposition to GnRH neurons. Treating OVX gilts with the progestin altrenogest decreased LH pulse frequency and reduced mean circulating concentrations of LH compared with OVX control gilts (P < 0.01), but the number of kisspeptin and NKB cells in the ARC did not differ between treatments. The neuroanatomical arrangement of kisspeptin, NKB, and NK3R within the porcine hypothalamus confirms they are positioned to stimulate GnRH and LH secretion in gilts, though differences with other species exist. Altrenogest suppression of LH secretion in the OVX gilt does not appear to involve decreased peptide expression of kisspeptin or NKB.

Effect of electroacupuncture on the kisspeptin system in a pubertal rat model of polycystic ovary syndrome.

OBJECTIVE: To explore the effects and mechanism of action of electroacupuncture (EA) in a rat model of pubertal polycystic ovary syndrome (PCOS). METHODS: Female offspring of Sprague-Dawley rats receiving dihydrotestosterone (DHT) during pregnancy (days 16-19), as a model of prenatal androgenization, were divided randomly into three groups: model group (M), EA group, and sham acupuncture (SA) group (n = 8 each). A normal (N) group comprising female offspring of healthy pregnant rats not receiving DHT (n = 8) was added. EA was administered at CV6 and bilateral SP6/ST36 with 2 Hz frequency and 2 mA intensity. SA consisted of superficial needling at different locations without electrical stimulation. RESULTS: EA improved the disturbed estrous cycles, while it could not be concluded that SA was effective in this respect. EA improved ovarian morphology including the number of corpora lutea and area of the ovary, whereas SA did not. However, both EA and SA attenuated the increased luteinizing hormone and decreased estradiol and gonadotropin-releasing hormone levels in the serum of PCOS model rats. Levels of testosterone, follicle-stimulating hormone, and progesterone did not significantly differ between groups. EA and SA alleviated the upregulation of kisspeptin protein and mRNA levels in the hypothalamus and kisspeptin protein level in the arcuate nucleus (ARC). No differences were found between groups in protein or mRNA expression of dynorphin (DYN) or neurokinin B (NKB) in the hypothalamus. Co-expression of kisspeptin, NKB, and DYN were observed in ARC. The GnRH level in the median eminence decreased and could be rescued by EA and SA. Intriguingly, kisspeptin levels in the granulosa cells of the ovary decreased in the model group and could be rescued by EA but not SA. Levels of kisspeptin, NKB, and DYN protein and mRNA in the ovary did not differ between any groups. CONCLUSION: Both EA and SA appeared to improve symptoms of PCOS at puberty by modulating the kisspeptin system in the hypothalamus. EA also had an effect on ovarian kisspeptin expression and a more comprehensive effect with respect to improving PCOS at puberty than SA.

Expression of genes for Kisspeptin (KISS1), Neurokinin B (TAC3), Prodynorphin (PDYN), and gonadotropin inhibitory hormone (RFRP) across natural puberty in ewes.

Expression of particular genes in hypothami of ewes was measured across the natural pubertal transition by in situ hybridization. The ewes were allocated to three groups (n = 4); prepubertal, postpubertal and postpubertally gonadectomized (GDX). Prepubertal sheep were euthanized at 20 weeks of age and postpubertal animals at 32 weeks. GDX sheep were also euthanized at 32 weeks, 1 week after surgery. Expression of KISS1, TAC3, PDYN in the arcuate nucleus (ARC), RFRP in the dorsomedial hypothalamus and GNRH1 in the preoptic area was quantified on a cellular basis. KISS1R expression by GNRH1 cells was quantified by double-label in situ hybridization. Across puberty, detectable KISS1 cell number increased in the caudal ARC and whilst PDYN cell numbers were low, numbers increased in the rostral ARC. TAC3 expression did not change but RFRP expression/cell was reduced across puberty. There was no change across puberty in the number of GNRH1 cells that expressed the kisspeptin receptor (KISS1R). GDX shortly after puberty did not increase expression of any of the genes of interest. We conclude that KISS1 expression in the ARC increases during puberty in ewes and this may be a causative factor in the pubertal activation of the reproductive axis. A reduction in expression of RFRP may be a factor in the onset of puberty, removing negative tone on GNRH1 cells. The lack of changes in expression of genes following GDX suggest that the effects of gonadal hormones may differ in young and mature animals.

Central Orexin A Affects Reproductive Axis by Modulation of Hypothalamic Kisspeptin/Neurokinin B/Dynorphin Secreting Neurons in the Male Wistar Rats.

It is an established fact that orexin plays an important role in regulating the reproductive axis and the secretions of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH). However, its precise cellular and molecular mechanisms are not fully recognized. Accordingly, the aim of the present study is to find out whether the central injection of orexin A (OXA) and its antagonists, SB-334867 (as orexin receptor antagonist 1; OX1RA) and JNJ-10397049 (as orexin receptor antagonist 2; OX2RA), either alone or in combination, can leave any impact on the reproductive axis (either hormonal or behavioral) in the male Wistar rats. Furthermore, in order to see whether OXA signals can be relayed through the pathway of kisspeptin/neurokinin B/dynorphin (known as KNDy neurons, a neural network which works upstream of GnRH neurons) or not, the relative gene expression of these neuropeptides were measured. Overall, the data from radioimmunoassay revealed that OXA significantly decreases the mean serum level of LH and testosterone and, in a similar vein, its antagonists neutralize this impact. Moreover, data from real-time quantitative PCR indicated that OXA has significantly reduced the hypothalamic expression of Gnrh. In this line, the gene expressions of Kisspeptin and Neurokinin b decreased. However, OXA antagonists neutralize this impact. Also, the expression of Dynorphin gene was upregulated by the following application of the OXA. The results of this study are related to the impact of orexin on the reproductive axis. It is recommended that KNDy neurons as the interneural pathway relay the information of orexin to the GnRH neurons.

The effects of chronic testosterone administration on hypothalamic gonadotropin-releasing hormone regulatory factors (Kiss1, NKB, pDyn and RFRP) and their receptors in female rats.

The effects of androgens on gonadotropin-releasing hormone (GnRH) secretion in females have not been fully established. To clarify the direct effects of androgens on hypothalamic reproductive factors, we evaluated the effects of chronic testosterone administration on hypothalamic GnRH regulatory factors in ovariectomized (OVX) female rats. Both testosterone and estradiol reduced the serum luteinizing hormone levels of OVX female rats, indicating that, as has been found for estrogen, testosterone suppresses GnRH secretion via negative feedback. Similarly, the administration of testosterone or estradiol suppressed the hypothalamic mRNA levels of kisspeptin and neurokinin B, both of which are positive regulators of GnRH, whereas it did not affect the hypothalamic mRNA levels of the kisspeptin receptor or neurokinin-3 receptor. On the contrary, the administration of testosterone, but not estradiol, suppressed the hypothalamic mRNA expression of prodynorphin, which is a negative regulator of GnRH. The administration of testosterone did not alter the rats' serum estradiol levels, indicating that testosterone's effects on hypothalamic factors might be induced by its androgenic activity. These findings suggest that as well as estrogen, androgens have negative feedback effects on GnRH in females and that the underlying mechanisms responsible for these effects are similar, but do not completely correspond, to the mechanisms underlying the effects of estrogen on GnRH.

Engineering a gene silencing viral construct that targets the cat hypothalamus to induce permanent sterility: An update.

The intent of this contribution is to provide an update of the progress we have made towards developing a method/treatment to permanently sterilize cats. Our approach employs two complementary methodologies: RNA interference (RNAi) to silence genes involved in the central control of reproduction and a virus-based gene therapy system intended to deliver RNAi selectively to the hypothalamus (where these genes are expressed) via the systemic administration of modified viruses. We selected the hypothalamus because it contains neurons expressing Kiss1 and Tac3, two genes essential for reproduction and fertility. We chose the non-pathogenic adeno-associated virus (AAV) as a vector whose tropism could be modified to target the hypothalamus. The issues that must be overcome to utilize this vector as a delivery vehicle to induce sterility include modification of the wild-type AAV to target the hypothalamic region of the brain with a simultaneous reduction in targeting of peripheral tissues and non-hypothalamic brain regions, identification of RNAi targets that will effectively reduce the expression of Kiss1 and Tac3 without off-target effects, and determination if neutralizing antibodies to the AAV serotype of choice are present in cats. Successful resolution of these issues will pave the way for the development of a powerful tool to induce the permanent sterility in cats.

Epigenetic regulation of puberty via Zinc finger protein-mediated transcriptional repression.

In primates, puberty is unleashed by increased GnRH release from the hypothalamus following an interval of juvenile quiescence. GWAS implicates Zinc finger (ZNF) genes in timing human puberty. Here we show that hypothalamic expression of several ZNFs decreased in agonadal male monkeys in association with the pubertal reactivation of gonadotropin secretion. Expression of two of these ZNFs, GATAD1 and ZNF573, also decreases in peripubertal female monkeys. However, only GATAD1 abundance increases when gonadotropin secretion is suppressed during late infancy. Targeted delivery of GATAD1 or ZNF573 to the rat hypothalamus delays puberty by impairing the transition of a transcriptional network from an immature repressive epigenetic configuration to one of activation. GATAD1 represses transcription of two key puberty-related genes, KISS1 and TAC3, directly, and reduces the activating histone mark H3K4me2 at each promoter via recruitment of histone demethylase KDM1A. We conclude that GATAD1 epitomizes a subset of ZNFs involved in epigenetic repression of primate puberty.

Molecular profiling of postnatal development of the hypothalamus in female and male rats.

Reproductive function is highly dynamic during postnatal developmental. Here, we performed molecular profiling of gene expression patterns in the hypothalamus of developing male and female rats to identify which genes are sexually dimorphic, to gain insight into a more complex network of hypothalamic genes, and to ascertain dynamic changes in their relationships with one another and with sex steroid hormones during development. Using a low-density PCR platform, we quantified mRNA levels in the preoptic area (POA) and medial basal hypothalamus (MBH), and assayed circulating estradiol, testosterone, and progesterone at six ages from birth through adulthood. Numerous genes underwent developmental change, particularly postnatal increases, decreases, or peaks/plateaus at puberty. Surprisingly, there were few sex differences; only Esr1, Kiss1, and Tac2 were dimorphic (higher in females). Cluster analysis of gene expression revealed sexually dimorphic correlations in the POA but not the MBH from P30 (Postnatal Day 30) to P60. Hormone measurements showed few sex differences in developmental profiles of estradiol; higher levels of progesterone in females only after P30; and a developmental pattern of testosterone with a nadir at P30 followed by a dramatic increase through P60 (males). Furthermore, bionetwork analysis revealed that hypothalamic gene expression profiles and their relationships to hormones undergo dynamic developmental changes that differ considerably from adults. These data underscore the importance of developmental stage in considering the effects of hormones on the regulation of neuroendocrine genes in the hypothalamus. Moreover, the finding that few neuroendocrine genes are sexually dimorphic highlights the need to consider postnatal development from a network approach that allows assessment of interactions and patterns of expression.

High-fat diet increases LH pulse frequency and kisspeptin-neurokinin B expression in puberty-advanced female rats.

To investigate whether the advancement of puberty in response to high-fat diet (HFD) results from a concomitant increase in LH pulse frequency and kisspeptin (Kiss1) and neurokinin B (NKB) signaling in the hypothalamus, blood samples were collected on postnatal day (pnd) 28, 32, or 36 for LH measurement and vaginal opening monitored as a marker of puberty in female rats fed with HFD or standard chow from weaning. Quantitative RT-PCR was used to determine Kiss1 and kisspeptin receptor (Kiss1r) mRNA levels in brain punches of the medial preoptic area and the arcuate nucleus (ARC), and NKB and NKB receptor (NK3R) mRNA levels in the ARC. There was a gradual increase in LH pulse frequency from pnd 28, reaching significance by pnd 36 in control diet-fed rats. The advancement of puberty by approximately 6 d (average pnd 34) in rats fed HFD was associated with an earlier onset of the higher LH pulse frequency that was already extant on pnd 28. The increased levels of expression of Kiss1 in the medial preoptic area and ARC, and NKB in the ARC, associated with pubertal onset were similarly advanced in HFD-fed rats. These data suggest that the earlier accelerated GnRH pulse generator frequency and advanced puberty with obesogenic diets might be associated with premature up-regulation of kisspeptin and NKB signaling in the hypothalamus of the female rat.

Fasting reduces the kiss1 mRNA levels in the caudal hypothalamus of gonadally intact adult female rats.

Kisspeptin, which is the product of the kiss1 gene and its receptor kiss1r, have emerged as the essential gatekeepers of reproduction. The present study used gonadally intact female rats to evaluate fasting-induced suppression of the KiSS-1 system of anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) under normal physiological conditions. Starting on the day of estrous, one group of rats was subjected to 72 h of food deprivation, while the other group of rats was able to continue feeding ad libitum. The length of the estrous cycle was significantly longer in the food-deprived rats as compared to the feeding rats. At the end of the 72-h food deprivation period, all of the food-deprived rats were at the diestrous phase, with their serum concentrations of LH and leptin significantly lower than that observed in the feeding rats. In addition, as compared to the feeding rats, the expression levels of kiss1 mRNA were significantly lower in the food-deprived rats in the posterior hypothalamic block, which contained the ARC, but not in the anterior hypothalamic block, which contain the AVPV. However, both the kiss1r mRNA expression levels in the anterior and posterior hypothalamic blocks and the neurokinin B and neurokinin 3 receptor mRNA expression levels in the posterior hypothalamic block were not significantly different between the feeding and food-deprived rats. Thus, lower kiss1 mRNA levels in the ARC appear to be responsible for the fasting-induced inhibition of gonadotrophin secretion and subsequent prolongation of the estrous cycle.

Autocrine regulation of human sperm motility by tachykinins.

BACKGROUND: We examined the presence and function of tachykinins and the tachykinin-degrading enzymes neprilysin (NEP) and neprilysin-2 (NEP2) in human spermatozoa. METHODS: Freshly ejaculated semen was collected from forty-eight normozoospermic human donors. We analyzed the expression of substance P, neurokinin A, neurokinin B, hemokinin-1, NEP and NEP2 in sperm cells by reverse-transcriptase polymerase chain reaction (RT-PCR), western blot and immunocytochemistry assays and evaluated the effects of the neprilysin and neprilysin-2 inhibitor phosphoramidon on sperm motility in the absence and presence of tachykinin receptor-selective antagonists. Sperm motility was measured using WHO procedures or computer-assisted sperm analysis (CASA). RESULTS: The mRNAs of the genes that encode substance P/neurokinin A (TAC1), neurokinin B (TAC3), hemokinin-1 (TAC4), neprilysin (MME) and neprilysin-2 (MMEL1) were expressed in human sperm. Immunocytochemistry studies revealed that tachykinin and neprilysin proteins were present in spermatozoa and show specific and differential distributions. Phosphoramidon increased sperm progressive motility and its effects were reduced in the presence of the tachykinin receptor antagonists SR140333 (NK1 receptor-selective) and SR48968 (NK2 receptor-selective) but unmodified in the presence of SR142801 (NK3 receptor-selective). CONCLUSION: These data show that tachykinins are present in human spermatozoa and participate in the regulation of sperm motility. Tachykinin activity is regulated, at least in part, by neprilysins.

Menopause and the human hypothalamus: evidence for the role of kisspeptin/neurokinin B neurons in the regulation of estrogen negative feedback.

Menopause is characterized by depletion of ovarian follicles, a reduction of ovarian hormones to castrate levels and elevated levels of serum gonadotropins. Rather than degenerating, the reproductive neuroendocrine axis in postmenopausal women is intact and responds robustly to the removal of ovarian hormones. Studies in both human and non-human primates provide evidence that the gonadotropin hypersecretion in postmenopausal women is secondary to increased gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus. In addition, menopause is accompanied by hypertrophy of neurons in the infundibular (arcuate) nucleus expressing KiSS-1, neurokinin B (NKB), substance P, dynorphin and estrogen receptor alpha (ERalpha) mRNA. Ovariectomy in experimental animals induces nearly identical findings, providing evidence that these changes are a compensatory response to ovarian failure. The anatomical site of the hypertrophied neurons, as well as the extensive data implicating kisspeptin, NKB and dynorphin in the regulation of GnRH secretion, provide compelling evidence that these neurons are part of the neural network responsible for the increased levels of serum gonadotropins in postmenopausal women. We propose that neurons expressing KiSS-1, NKB, substance P, dynorphin and ERalpha mRNA in the infundibular nucleus play an important role in sex-steroid feedback on gonadotropin secretion in the human.

The effects of hormone replacement therapy on hypothalamic neuropeptide gene expression in a primate model of menopause.

Menopause is associated with increased neurokinin B (NKB) gene expression and decreased proopiomelanocortin (POMC) gene expression in the human hypothalamus. In the present study, young, ovariectomized cynomolgus monkeys were used in a model of menopause to examine the effects of hormone replacement therapy (HRT) on hypothalamic neuropeptide gene expression. A secondary goal was to determine whether HRT produces signs of estrogen toxicity in the primate hypothalamus by examining POMC neurons and microglial cells. In situ hybridization was performed using synthetic, radiolabeled, 48-base oligonucleotide probes. Alpha-napthyl butyrate esterase histochemistry was used to visualize microglial cells. Both estrogen and estrogen plus progesterone treatments produced a marked suppression of the number of infundibular neurons expressing NKB gene transcripts. In contrast, HRT had no effect on the POMC system of neurons or the number of microglial cells in the infundibular nucleus. These results provide strong support for the hypothesis that the increased NKB gene expression in the hypothalamus of postmenopausal women is secondary to estrogen withdrawal. Conversely, these data suggest that the dramatic decline in the numbers of neurons expressing POMC gene transcripts in older women is caused by factors other than ovarian failure. Finally, we found no evidence that HRT, in doses designed to mimic currently prescribed regimens, produces signs of estrogen toxicity in the primate infundibular nucleus.

Localization of neurons expressing substance P and neurokinin B gene transcripts in the human hypothalamus and basal forebrain.

In situ hybridization histochemistry was used to map the distribution of neurons expressing the substance P (SP) or neurokinin B (NKB) genes in the human hypothalamus and basal forebrain. Hypothalami from five adult males were frozen in isopentane at -30 degrees C and serially sectioned at 20 jm thickness. Every 20th section was hybridized with [35S]-labeled, 48-base synthetic cDNA probes that were complementary to either SP or NKB mRNAs. Slides were dipped into nuclear emulsion for visualization of mRNAs at the single-cell level. The location of labeled neurons (greater than x 5 background) was mapped by using an image-combining computer microscope system. A distinct and complementary distribution pattern of SP and NKB neurons was observed in the human hypothalamus and basal forebrain. NKB was the predominant tachykinin in the rostral hypothalamus, whereas SP mRNA predominated in the posterior hypothalamus. Numerous NKB neurons were identified in the magnocellular basal forebrain, the bed nucleus of stria terminalis, and the anterior hypothalamic area. Scattered NKB neurons were present in the infundibular and paraventricular nuclei, paraolfactory gyrus, posterior hypothalamic area, lateral division of the medial mammillary nucleus, and amygdala. Numerous neurons expressing SP mRNAs were identified in the premammillary, supramammillary, and medial mammillary nuclei; the posterior hypothalamic area; and the corpus striatum. Scattered SP neurons were also observed in the preoptic area; the infundibular, intermediate, dorsomedial, and ventromedial nuclei; the infundibular stalk; the amygdala; the bed nucleus of stria terminalis; and the paraolfactory gyrus. These studies provide the first description of the location of neurons that express tachykinin gene transcripts in the human hypothalamus.

Hypertrophy and increased gene expression of neurons containing neurokinin-B and substance-P messenger ribonucleic acids in the hypothalami of postmenopausal women.

We have previously described hypertrophy of neurons containing estrogen receptor mRNA in the infundibular nucleus of postmenopausal women. In the present investigation we identified peptide mRNAs in the hypertrophied neurons and determined whether postmenopausal neuronal hypertrophy was accompanied by changes in gene expression. In the first study in situ hybridization was performed on sections from hypothalami of postmenopausal women (n = 3) using synthetic 35S-labeled cDNA probes complementary to mRNAs encoding estrogen receptor, substance-P (SP), neurokinin-B (NKB), POMC, cholecystokinin, dynorphin, CRF, enkephalin, galanin, neuropeptide-Y, GH-releasing hormone, and tyrosine hydroxylase. Neuronal cross-sectional areas and cell densities were measured with the aid of a computer microscope system. Neurons labeled with the NKB and SP probes were comparable in size, morphology, and distribution to the hypertrophied neurons containing estrogen receptor mRNA. In contrast, neurons labeled with other cDNA probes were sparsely distributed (CRF and dynorphin), smaller in size (neuropeptide-Y, galanin, GH-releasing hormone, enkephalin, cholecystokinin, and POMC), or located anterior to the hypertrophied population (tyrosine hydroxylase). In the second study sections from hypothalami of premenopausal (n = 3) and postmenopausal (n = 3) women were incubated with cDNA probes complementary to SP or NKB mRNAs. The mean cross-sectional areas of postmenopausal infundibular neurons containing NKB and SP mRNAs increased to 194% and 176% of premenopausal values, respectively. The autoradiographic grain densities of infundibular neurons labeled with either probe were also significantly increased in the postmenopausal group. Finally, the numbers of labeled neurons/tissue increased 6-fold (SP) and 15-fold (NKB) in the postmenopausal infundibular nucleus. These data demonstrate that human menopause is associated with marked increases in hypothalamic NKB and SP gene expression. We propose that neurons containing estrogen receptor, SP, and NKB mRNAs participate in the hypothalamic circuitry regulating estrogen negative feedback in the human.

Neurokinin B and substance P genes are co-expressed in a subset of neurons in the rat habenula.

Colocalization of neurokinin B (NKB) and substance P (SP) mRNAs in neurons of the habenula was examined on thin, adjacent sections in the rat. Extensive colocalization was found in the medial habenula. In its dorsolateral part, most of the neurons contained both transcripts, with high levels of SP, while in the dorsomedial part fewer instances of colocalization were found. The ventral half could be divided into two parts, a dorsal part with most of the neurons containing both messages, having low levels of SP mRNA, and a ventral part with most of the cells containing only NKB mRNA. Cells in the medial habenula had low levels of NKB mRNA. These results suggest a structural and functional heterogeneity of the medial habenula.