Recent studies suggest that arcuate neurokinin B (NKB) neurons play a role in the regulation of gonadotropin secretion, but there is little information on the relationship between these neurons and the hypothalamic reproductive axis. In the present study, dual-label fluorescent immunohistochemistry was used to visualize the relationship between gonadotropin-releasing hormone (GnRH) neurons and either proNKB or NK3 receptor (NK3R) immunoreactivity. Immunocytochemistry was also combined with i.p. injections of the fluorescent retrograde tracer aminostilbamidine to determine whether arcuate neuroendocrine neurons expressed either proNKB or NK3R. A dense interweaving and close apposition of GnRH and proNKB-immunoreactive (ir) fibers was observed within the rat median eminence, where GnRH axons expressed NK3R immunoreactivity. These data provide morphological evidence that NKB neurons could influence GnRH secretion via interaction with NK3R in the rat median eminence. Colocalization of GnRH and NK3R was also identified in fiber tracts converging within the organum vasculosum of the lamina terminalis. In contrast, only a small number (16%) of GnRH-ir somata exhibited NK3R staining. ProNKB and NK3R-ir somata were identified within the arcuate nucleus, but none of these neurons were labeled by aminostilbamidine. Thus, we found no evidence that arcuate NKB neurons project to the primary capillary plexus of the portal system. Arcuate neuroendocrine neurons, however, were surrounded and closely apposed by proNKB-ir puncta and fibers. These data suggest that NKB neurons could indirectly influence anterior pituitary function by inputs to arcuate neuroendocrine neurons, but through a receptor other than NK3R. Our results provide an anatomic framework for putative interactions between NKB neurons and the hypothalamic reproductive axis.

Neurokinin B (NKB) and kisspeptin receptor signaling are essential components of the reproductive axis. A population of neurons resides within the arcuate nucleus of the rat that expresses NKB, kisspeptin, dynorphin, NK3 receptors and estrogen receptor α. Here we investigate the projections of these neurons using NKB-immunocytochemistry as a marker. First, the loss of NKBimmunoreactive (ir) somata and fibers was characterized after ablation of the arcuate nucleus by neonatal injections of monosodium glutamate. Second, biotinylated dextran amine was injected into the arcuate nucleus and anterogradely labeled NKB-ir fibers were identified using dual-labeled immunofluorescence. Four major projection pathways are described: 1) Local projections within the arcuate nucleus bilaterally, 2) Projections to the median eminence including the lateral palisade zone, 3) Projections to a periventricular pathway extending rostrally to multiple hypothalamic nuclei, the septal region and BNST and dorsally to the dorsomedial nucleus and 4) Projections to a ventral hypothalamic tract to the lateral hypothalamus and medial forebrain bundle. The diverse projections provide evidence that NKB/kisspeptin/dynorphin neurons could integrate the reproductive axis with multiple homeostatic, behavioral and neuroendocrine processes. Interestingly, anterograde tracttracing revealed NKB-ir axons originating from arcuate neurons terminating on other NKB-ir somata within the arcuate nucleus. Combined with previous studies, these experiments reveal a bilateral interconnected network of sex-steroid responsive neurons in the arcuate nucleus of the rat that express NKB, kisspeptin, dynorphin, NK3 receptors and ERα and project to GnRH terminals in the median eminence. This circuitry provides a mechanism for bilateral synchronization of arcuate NKB/ kisspeptin/dynorphin neurons to modulate the pulsatile secretion of GnRH. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2011 March 17. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptLoss of function mutations in neurokinin B (NKB), the neurokinin 3 (NK3) receptor (the primary NKB receptor) or the kisspeptin receptor (GPR54) result in hypogonadotrophic hypogonadism with absence of pubertal development (Seminara et al., 2003;de Roux et al., 2003;Topaloglu et al., 2009). These studies document an essential role of NKB and kisspeptin receptor signaling in the regulation of the human reproductive axis. A key element of this regulatory circuitry is a gro...

In this study, we examined chronic norepinephrine suppression of insulin secretion in sheep fetuses with placental insufficiency-induced intrauterine growth restriction (IUGR). Glucose-stimulated insulin secretion (GSIS) was measured with a square-wave hyperglycemic clamp in the presence or absence of adrenergic receptor antagonists phentolamine (alpha) and propranolol (beta). IUGR fetuses were hypoglycemic and hypoxemic and had lower GSIS responsiveness (P < or = 0.05) than control fetuses. IUGR fetuses also had elevated plasma norepinephrine (3,264 +/- 614 vs. 570 +/- 86 pg/ml; P < or = 0.05) and epinephrine (164 +/- 32 vs. 60 +/- 12 pg/ml; P < or = 0.05) concentrations. In control fetuses, adrenergic inhibition increased baseline plasma insulin concentrations (1.7-fold, P < or = 0.05), whereas during hyperglycemia insulin was not different. A greater (P < or = 0.05) response to adrenergic inhibition was found in IUGR fetuses, and the average plasma insulin concentrations increased 4.9-fold at baseline and 7.1-fold with hyperglycemia. Unlike controls, basal plasma glucose concentrations fell (P < or = 0.05) with adrenergic antagonists. GSIS responsiveness, measured by the change in insulin, was higher (8.9-fold, P < or = 0.05) in IUGR fetuses with adrenergic inhibition than controls (1.8-fold, not significant), showing that norepinephrine suppresses insulin secretion in IUGR fetuses. Strikingly, in IUGR fetuses, adrenergic inhibition resulted in a greater GSIS responsiveness, because beta-cell mass was 56% lower and the maximal stimulatory insulin response tended (P < 0.1) to be higher than controls. This persistent norepinephrine suppression appears to be partially explained by higher mRNA concentrations of adrenergic receptors alpha(1D), alpha(2A), and alpha(2B) in a cohort of fetuses that were naïve to the antagonists. Therefore, norepinephrine suppression of insulin secretion was maintained, in part, by upregulating adrenergic receptor expression, but the beta-cells also appeared to compensate with enhanced GSIS. These findings may begin to explain why IUGR infants have a propensity for increased glucose requirements if norepinephrine is suddenly decreased after birth.

Islet replacement is a promising therapy for treating Diabetes Mellitus, but the supply of donor tissue for transplantation is limited. To overcome this limitation, endocrine tissue can be expanded, but this requires an understanding of normal developmental processes that regulate islet formation. In this study we compare pancreas development in the sheep and human, and provide evidence that an epithelial-mesenchymal transition (EMT) is involved in β-cell differentiation and islet formation. Transcription factors know to regulate pancreas formation, Pdx1, Neurogenin 3, Nkx2.2 and Nkx6.1, were expressed in the appropriate spatial and temporal pattern to coordinate pancreatic bud outgrowth and direct endocrine cell specification in the sheep. Immunofluorescence staining of the developing pancreas was used to co-localize insulin and epithelial proteins (cytokeratin, E-cadherin, and β-catenin) or insulin and a mesenchymal protein (vimentin). In the sheep, individual β-cells become insulin-positive in the progenitor epithelium, then lose epithelial characteristics, and migrate out of the epithelial layer to form islets. As β-cells exit the epithelial progenitor cell layer they acquire mesenchymal characteristics, shown by their acquisition of vimentin. In situ hybridization expression analysis of the Snail family members of transcriptional repressors (Snail-1, -2, and -3) showed that each of the Snail genes was expressed in the ductal epithelium during development, and Snail-1 and 2 were co-expressed with insulin. Our findings provide strong evidence that the movement of β-cells from the pancreatic ductal epithelium involves an EMT.

Key pointsr To investigate loss of skeletal muscle mass in intrauterine growth-restricted (IUGR) fetuses near term, which may result from myoblast dysfunction, we examined semitendinosus myofibre and myoblast morphology in placental insufficiency-induced IUGR sheep fetuses; we also isolated and cultured IUGR fetal myoblasts to determine whether reduced rates of proliferation were due to intrinsic cellular defects or extrinsic factors associated with serum.r Using tests for myogenin and pax7 to identify differentiated and undifferentiated fetal myoblasts, respectively, we found that myofibre area and percentage of myogenin-positive nuclei were less in IUGR fetal semitendinosus muscles than in controls, but myofibre density and percentage of pax7-positive nuclei were not different.r The percentage of pax7-positive cells that expressed proliferating cellular nuclear antigen was less in IUGR semitendinosus muscles than in controls, while in myoblasts isolated from fetal sheep and replicated and differentiated in culture, IUGR fetal myoblasts proliferated at slower rates than control myoblasts, under identical culture conditions, but the ability to differentiate was similar between treatments.r Media supplemented with IUGR serum decreased replication rates in both IUGR and control myoblasts compared to media supplemented with control fetal serum.r These findings show that myoblasts proliferate at slower rates in IUGR fetuses due to a combination of intrinsic cellular characteristics and extrinsic serum factors; the intrinsic defects may explain reduced skeletal muscle mass observed in IUGR newborn children and adults.Abstract Intrauterine growth restriction (IUGR) reduces skeletal muscle mass in fetuses and offspring. Our objective was to determine whether myoblast dysfunction due to intrinsic cellular deficiencies or serum factors reduces myofibre hypertrophy in IUGR fetal sheep. At 134 days, IUGR fetuses weighed 67% less (P < 0.05) than controls and had smaller (P < 0.05) carcasses and semitendinosus myofibre areas. IUGR semitendinosus muscles had similar percentages of pax7-positive nuclei and pax7 mRNA but lower (P < 0.05) percentages of myogenin-positive nuclei (7 ± 2% and 13 ± 2%), less myoD and myogenin mRNA, and fewer (P < 0.05) proliferating myoblasts (PNCA-positive-pax7-positive) than controls (44 ± 2% vs. 52 ± 1%). Primary myoblasts were isolated from hindlimb muscles, and after 3 days in growth media (20% fetal bovine serum, FBS), myoblasts from IUGR fetuses had 34% fewer (P < 0.05) myoD-positive cells than controls and replicated 20% less (P < 0.05) during a 2 h BrdU pulse. IUGR myoblasts also replicated less (P < 0.05) than controls during a BrdU pulse after 3 days in media containing 10% control or IUGR fetal sheep serum (FSS). Both myoblast types replicated less (P < 0.05) with IUGR FSS-supplemented media compared to control FSS-supplemented media. In differentiation-promoting media (2% FBS), IUGR and control myoblasts had similar percentages of myogenin-positive nuclei after 5 days and formed similar...