Description: Puberty onset in female sheep is marked by a decrease in estradiol-negative feedback, allowing for the increase in GnRH and LH pulses that heralds the first ovulation. Based on recent genetic studies in humans, two possible neuropeptides that could promote puberty onset are kisspeptin and neurokinin B (NKB). Our first experiment determined whether the NKB agonist, senktide, could stimulate LH secretion in prepubertal ewes. A second study used prepubertal and postpubertal ewes that were intact or ovariectomized (OVX) to test the hypothesis that expression of kisspeptin and NKB in the arcuate nucleus increased postpubertally. For comparison, kisspeptin and NKB expression in age-matched intact, and castrated males were also examined. In experiment 1, the percentage of ewes showing an LH pulse immediately after injection of senktide (100 μg, 60%; 500 μg, 100%) was greater than that for water-injected controls (experiment 1a, 25%; experiment 1b, 20%). In experiment 2, kisspeptin-positive cell numbers in the arcuate nucleus increased after puberty in intact females and were increased by OVX in prepubertal but not postpubertal ewes. Changes in kisspeptin cell numbers were paralleled by changes in kisspeptin-close contacts onto GnRH neurons in the medial preoptic area. NKB cell numbers did not differ significantly between intact prepubertal and postpubertal ewes but increased with OVX in both age groups. NKB fiber immunoreactivity was greater in postpubertal than in prepubertal intact ewes. In age-matched males, kisspeptin and NKB cell numbers increased with castration, but decreased with age. These results support the hypothesis that kisspeptin is a gatekeeper to female ovine puberty and raise the possibility that NKB may also play a role, albeit through different means.

Description: Although several lines of evidence have indicated that menopause is associated with increased susceptibility to neurological disorders, the mechanisms involved in this phenomenon remain to be elucidated. Because neuroinflammation is a common feature of a number of brain diseases, we hypothesized that the cessation of ovarian functions and the consequent decrease in estrogen receptor (ER)-mediated antiinflammatory activity may represent a trigger for postmenopausal brain dysfunctions. The aim of the present study was to investigate the effects of aging and surgical menopause on the activity of ER in neuroinflammation. The present study shows that ER genes are expressed in the hippocampus, but ER transcriptional activity decreases significantly beginning at 12 months of age in intact and ovariectomized mice. With ovariectomy, we observe an age-dependent accumulation of mRNA encoding inflammatory mediators ( e.g . TNFα, IL1β, and macrophage inflammatory protein-2) and changes in the morphology of astroglia and microglia. In addition, we show that aging itself is coupled with an exaggerated response to acute inflammatory stimuli with a major accumulation of TNFα, IL1β, macrophage inflammatory protein-2, and macrophage chemoattractant protein-1 mRNA in response to lipopolysaccharide administration. The response to acute inflammatory stimuli appears to be differentially modulated by the duration of hormone deprivation in 12-month-old mice. Taken together, the present results show that aging is associated with decreased ER activity, despite continuous ER synthesis, and that age-dependent neuroinflammation is strongly influenced by hormone deprivation.

Description: Age-related depletion of estrogens and androgens is associated with an increase in Alzheimer's disease (AD) brain pathology and diminished cognitive function. Here we investigated AD-associated molecular and cellular changes in brains of aged hypogonadal ( hpg ) male and female mice. hpg Mice have a spontaneous, inactivating genetic mutation in the GnRH gene resulting in life-long deficiency of gonadotropins and gonadal sex hormones. Western blot analysis revealed low levels of amyloid precursor protein and high levels of presenilin 1, amyloid precursor protein C-terminal fragment, and β-amyloid 42 in brains of aged male, but not female, hpg mice. Changes were confined to the hippocampus and were not evident in the cerebellum or other brain tissues. Male hpg mice tended to have lower levels of IL-1β protein than male littermate controls. Immunohistochemical staining of the basal forebrain revealed that male hpg mice had lower choline acetyltransferase levels per neuron compared with controls. These AD-like changes specific to male hpg mice supports a link between androgen depletion and the development of AD pathology.

Description: Although studies in vitro and in hypothyroid animals show that thyroid hormone can, under some circumstances, modulate the actions of low-density lipoprotein (LDL) receptors, the mechanisms responsible for thyroid hormone's lipid-lowering effects are not completely understood. We tested whether LDL receptor (LDLR) expression was required for cholesterol reduction by treating control and LDLR-knockout mice with two forms of thyroid hormone T 3 and 3,5-diiodo- l -thyronine. High doses of both 3,5-diiodo- l -thyronine and T 3 dramatically reduced circulating total and very low-density lipoprotein/LDL cholesterol (~70%) and were associated with reduced plasma T 4 level. The cholesterol reduction was especially evident in the LDLR-knockout mice. Circulating levels of both apolipoprotein B (apo)B48 and apoB100 were decreased. Surprisingly, this reduction was not associated with increased protein or mRNA expression of the hepatic lipoprotein receptors LDLR-related protein 1 or scavenger receptor-B1. Liver production of apoB was markedly reduced, whereas triglyceride production was increased. Thus, thyroid hormones reduce apoB lipoproteins via a non-LDLR pathway that leads to decreased liver apoB production. This suggests that drugs that operate in a similar manner could be a new therapy for patients with genetic defects in the LDLR.

Description: RCAN1 is a chromosome 21 gene that controls secretion in endocrine cells, regulates mitochondrial function, and is sensitive to oxidative stress. Regulator of calcineurin 1 (RCAN1) is also an endogenous inhibitor of the protein phosphatase calcineurin, the inhibition of which leads to hypoinsulinemia and diabetes in humans and mice. However, the presence or the role of RCAN1 in insulin-secreting β-cells and its potential role in the pathogenesis of diabetes is unknown. Hence, the aim of this study is to investigate the presence of RCAN1 in β-cells and identify its role in β-cell function. RCAN1 is expressed in mouse islets and in the cytosol of pancreatic β-cells. We find RCAN1 is a glucose-responsive gene with a 1.5-fold increase in expression observed in pancreatic islets in response to chronic hyperglycemia. The overexpression of the human RCAN1.1 isoform in mice under the regulation of its endogenous promoter causes diabetes, age-associated hyperglycemia, reduced glucose tolerance, hypoinsulinemia, loss of β-cells, reduced β-cell insulin secretion, aberrant mitochondrial reactive oxygen species production, and the down-regulation of key β-cell genes. Our data therefore identifies a novel molecular link between the overexpression of RCAN1 and β-cell dysfunction. The glucose-responsive nature of RCAN1 provides a potential mechanism of action associated with the β-cell dysfunction observed in diabetes.

Description: A low-grade proinflammatory state contributes to the metabolic syndrome (MS). Adiponectin (ApN), which is reduced in the MS, has emerged as a master regulator of inflammation/immunity. We wanted to identify whether microRNAs (miRNAs) may mediate the antiinflammatory action of ApN on adipose tissue (AT). miRNA expression profiling was performed in mice overexpressing ApN specifically in AT and in wild-type controls. The role of specific miRNAs was analyzed by gain- or loss-of function approaches in 3T3-F442A (pre)-adipocytes and in de novo AT formed from engineered 3T3-F442A preadipocytes transplanted in nude mice. miRNA expression was compared in the omental AT of lean and obese subjects. The expression of miR532-5p and miR1983 was down-regulated, whereas that of miR883b-5p and miR1934 was up-regulated in AT of mice overexpressing ApN specifically in AT. We focused on miR883b-5p identified by computational analysis as being involved in inflammatory pathways. miR883b-5p overexpression down-regulated the lipopolysaccharide-binding protein (LBP) in 3T3-F442A cells, whereas miR883b-5p blockade had reverse effects. LBP aids in lipopolysaccharide binding to Toll-like receptor-4. miR883b-5p blockade also abolished the protective effects of ApN on proinflammatory adipokine induction. These data were recapitulated in the de novo AT in which miR883b-5p silencing induced LBP production and tissue inflammation. Eventually miR883b-5p expression was down-regulated in AT of obese subjects. We identified several novel miRNAs that are regulated by ApN in AT in vivo . miR883b-5p, which is up-regulated by ApN represses LBP and Toll-like receptor-4 signaling, acting therefore as a major mediator of the antiinflammatory action of ApN. These novel miRNAs may open new therapeutic perspectives for the MS.

Description: Principal limitation of cell therapy is cell loss after transplantation because of the interplay between ischemia, inflammation, and apoptosis. We investigated the mechanism of preconditioning of mesenchymal stem cells (MSCs) with oxytocin (OT), which has been proposed as a novel strategy for enhancing therapeutic potential of these cells in ischemic heart. In this study, we demonstrate that rat MSCs express binding sites for OT receptor and OT receptor transcript and protein as detected by RT-PCR and immunofluorescence, respectively. In response to OT (10 –10 to 10 –6 m ) treatment, MSCs respond with rapid calcium mobilization and up-regulation of the protective protein kinase B (PKB or Akt) and phospho-ERK1/2 proteins. In OT-stimulated cells, phospho-Akt accumulates intracellularly close to the mitochondrial marker cytochrome c oxidase subunit 4. Functional analyses reveal the involvement of Akt/ERK1/2 pathways in cell proliferation, migration, and protection against the cytotoxic and apoptotic effects of hypoxia and serum deprivation. In addition, OT preconditioning increases MSC glucose uptake. Genes with angiogenic, antiapoptotic, and cardiac antiremodeling properties, such as heat shock proteins (hsps) HSP27, HSP32, HSP70, vascular endothelial growth factor, thrombospondin, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, TIMP-3, and matrix metalloproteinase-2, were also up-regulated upon OT exposure. Moreover, coculture with OT-preconditioned MSC reduces apoptosis, as measured using terminal transferase dUTP nick end labeling assay in newborn rat cardiomyocytes exposed to hypoxia and reoxygenation. In conclusion, these results indicate that OT treatment evokes MSC protection through both intrinsic pathways and secretion of cytoprotective factors. Ex vivo cellular treatment with OT represents an attractive strategy aimed to maximize the biological and functional properties of effector cells.

Description: Maternal exposure to increased synthetic glucocorticoids (GC) during pregnancy is known to disturb fetal development and increase the risk of long-term disease. Maternal exposure to elevated levels of natural GC is likely to be common yet is relatively understudied. The placenta plays an important role in regulating fetal exposure to maternal GC but is itself vulnerable to maternal insults. This study uses a mouse model of maternal corticosterone (Cort) exposure to investigate its effects on the developing placenta. Mice were treated with Cort (33 μg/kg·h) for 60 h starting at embryonic d 12.5 (E12.5) before collection of placentas at E14.5 and E17.5. Although Cort exposure did not affect fetal size, placentas of male fetuses were larger at E17.5 in association with changes in placental Igf2 . This increase in size was associated with an increase in placental thickness and an increase in placental junctional zone volume. Placentas from female fetuses were of normal size and had no changes in growth factor mRNA levels. The expression of the protective enzyme 11β-hydroxysteroid dehydrogenase type 2 was increased at E14.5 but was decreased in males at E17.5. In contrast, the expression of Nr3c1 (which encodes the GC receptor) was increased during the Cort exposure and remained elevated at E17.5 in the placentas of male fetuses. Our study has shown that maternal Cort exposure infers a sex-specific alteration to normal placental growth and growth factor expression, thus further adding to our understanding of the mechanisms of male dominance of programmed disease.

Description: Endometriosis is the estrogen-dependent growth of endometrial tissue outside the uterus. Endometriosis has an effect on the eutopic endometrium; however, the nature of the cellular or molecular signal from the lesion to the uterus is unknown. Here we demonstrate that cells migrate from endometriosis to eutopic endometrium. Experimental endometriosis was established by transplanting endometrial tissue from green fluorescent protein (GFP) mice to the peritoneal cavity of DS-Red mice. Immunofluorescence (IF) identified cells from the ectopic lesions in the uterus. The eutopic endometrial cells were sorted by fluorescence activated cell sorting, and the GFP + /DS-Red – population was characterized using microarray analysis. The results of cell sorting as well as the array results were confirmed by quantitative PCR and IF. GFP + /DS-red – /Cd45 – cells were identified in the eutopic endometrium of mice with experimental endometriois (~1.8%) and not in controls. Global gene expression profiling of these cells showed absence of leukocyte and increased expression of pan-epithelial markers in the uterine GFP + cells. Moreover, GFP + cells showed up-regulation of Wnt7A expression and 17 other genes associated with the Wingless pathway. Several genes that are associated with epithelial-to-mesenchymal transition were also highly differentially expressed in GFP + cells. IF confirmed the presence of the GFP + /CD45 – /Wnt7a + /cytokeritin + cells in the endometrium of endometriotic animals, and not in controls. Cells from endometriotic lesions are capable of migrating to the eutopic endometrium. The ectopic expression of Wnt7A suggests a possible mechanism by which ectopic lesions affect the eutopic endometrium and interfere with embryo implantation and fertility.

Description: Polycystic ovarian syndrome (PCOS) is a heterogeneous syndrome associated with follicle growth arrest, minimal granulosa cell proliferation, dysregulated sex hormone profile, hyperthecosis, and insulin resistance. Using a 5α-dihydrotestosterone (DHT)-induced rat model that recapitulates the reproductive and metabolic phenotypes of human PCOS, we have examined the steroidogenic capability of granulosa cells from DHT-treated rats. Gene expression of several key steroidogenic enzymes including p450 side-chain cleavage enzyme (p450scc), aromatase, steroidogenic acute regulatory protein, hydroxysteroid dehydrogenase-17β, and hydroxysteroid dehydrogenase-3β were markedly lower in DHT-treated rats than the controls, although the responsiveness of their granulosa cells to FSH was higher. Expression of the adipokine chemerin and its receptor, chemokine receptor-like 1, was evident in control and DHT-treated rats, with significantly higher ovarian mRNA abundances and protein contents of chemerin and its receptor. Recombinant chemerin decreases basal estradiol secretion in granulosa cells from DHT-treated rats. When the inhibitory role of chemerin on steroidogenesis was further examined in vitro , chemerin suppressed FSH-induced progesterone and estradiol secretion in cultured preantral follicles and granulosa cells. Chemerin also inhibits FSH-induced aromatase and p450scc expression in granulosa cells. Overexpression of nuclear receptors NR5a1 and NR5a2 promotes p450scc and aromatase expression, respectively, which is suppressed by chemerin. These findings suggest that chemerin is a novel negative regulator of FSH-induced follicular steroidogenesis and may contribute to the pathogenesis of PCOS.