H3K9me3 (histone H3 modified with tri-methylation at lysine 9) is a hallmark of transcriptional silencing and heterochromatin. However, its global effects on the genome, including euchromatin, are less well understood. Here we develop Formaldehyde-Assisted Identification of Regulatory Elements (FAIRE) for C. elegans to examine the chromatin configuration of mutants that lack virtually all H3K9me3, while leaving H3K9me1 and H3K9me2 intact. We find that nucleosomes are mildly disrupted, and levels of H3K9me2 and H3K27me3 rise in mutant embryos. In addition to these expected changes, the most dramatic change occurs in euchromatin: many regions encompassing transcription start sites (TSSs) gain an average of two nucleosomes in mutants. The affected regions normally lack H3K9me3, revealing a locus non-autonomous role for H3K9me3. Affected TSSs are associated with genes that are active in epithelia and muscles, and implicated in development, locomotion, morphogenesis and transcription. Mutant embryos develop normally under ideal laboratory conditions but die when challenged, with defects in morphogenesis and development. Our findings reveal that H3K9me3 protects transcription start sites within euchromatin from nucleosome deposition. These results may be relevant to mammals, where diseases that disrupt the nuclear lamina and heterochromatin can alter epithelial and muscle gene expression.

Study question Do naturally occurring, hyperandrogenic (≥1 SD of population mean testosterone, T) female rhesus monkeys exhibit traits typical of women with polycystic ovary syndrome (PCOS)? Summary answer Hyperandrogenic female monkeys exhibited significantly increased serum levels of androstenedione (A4), 17-hydroxyprogesterone (17-OHP), estradiol (E2), LH, antimullerian hormone (AMH), cortisol, 11-deoxycortisol and corticosterone, as well as increased uterine endometrial thickness and evidence of reduced fertility, all traits associated with PCOS. What is known already Progress in treating women with PCOS is limited by incomplete knowledge of its pathogenesis and the absence of naturally occurring PCOS in animal models. A female macaque monkey, however, with naturally occurring hyperandrogenism, anovulation and polyfollicular ovaries, accompanied by insulin resistance, increased adiposity and endometrial hyperplasia, suggests naturally occurring origins for PCOS in nonhuman primates. Study design, size, duration As part of a larger study, circulating serum concentrations of selected pituitary, ovarian and adrenal hormones, together with fasted insulin and glucose levels, were determined in a single, morning blood sample obtained from 120 apparently healthy, ovary-intact, adult female rhesus monkeys (Macaca mulatta) while not pregnant or nursing. The monkeys were then sedated for somatometric and ultrasonographic measurements. Participants/materials, setting, methods Female monkeys were of prime reproductive age (7.2 ± 0.1 years, mean ± SEM) and represented a typical spectrum of adult body weight (7.4 ± 0.2 kg; maximum 12.5, minimum 4.6 kg). Females were defined as having normal (n = 99) or high T levels (n = 21; ≥1 SD above the overall mean, 0.31 ng/ml). Electronic health records provided menstrual and fecundity histories. Steroid hormones were determined by tandem LC-MS-MS; AMH was measured by enzymeimmunoassay; LH, FSH and insulin were determined by radioimmunoassay; and glucose was read by glucose meter. Most analyses were limited to 80 females (60 normal T, 20 high T) in the follicular phase of a menstrual cycle or anovulatory period (serum progesterone <1 ng/ml). Main results and the role of chance Of 80 monkeys, 15% (n = 12) exhibited classifiable PCOS-like phenotypes. High T females demonstrated elevations in serum levels of LH (P < 0.036), AMH (P < 0.021), A4 (P < 0.0001), 17-OHP (P < 0.008), E2 (P < 0.023), glucocorticoids (P < 0.02-0.0001), the serum T/E2 ratio (P < 0.03) and uterine endometrial thickness (P < 0.014) compared to normal T females. Within the high T group alone, anogenital distance, a biomarker for fetal T exposure, positively correlated (P < 0.015) with serum A4 levels, while clitoral volume, a biomarker for prior T exposure, positively correlated (P < 0.002) with postnatal age. Only high T females demonstrated positive correlations between serum LH, and both T and A4. Five of six (83%) high T females with serum T ≥2 SD above T mean (0.41 ng/ml) did not produce live offspring. Large scale data N/A. Limitations, reasons for caution This is an initial study of a single laboratory population in a single nonhuman primate species. While two biomarkers suggest lifelong hyperandrogenism, phenotypic expression during gestation, prepuberty, adolescence, mid-to-late reproductive years and postmenopause has yet to be determined. Wider implications of the findings Characterizing adult female monkeys with naturally occurring hyperandrogenism has identified individuals with high LH and AMH combined with infertility, suggesting developmental linkage among traits with endemic origins beyond humans. PCOS may thus be an ancient phenotype, as previously proposed, with a definable pathogenic mechanism(s). Study funding/competing interest(s) Funded by competitive supplement to P51 OD011106 (PI: Mallick), by P50 HD028934 (PI: Marshall) and by P50 HD044405 (PI: Dunaif). The authors have no potential conflicts of interest.

Pioneer transcription factors initiate cell-fate changes by binding to silent target genes. They are among the first factors to bind key regulatory sites and facilitate chromatin opening. Here, we identify an additional role for pioneer factors. In early Caenorhabditis elegans foregut development, the pioneer factor PHA-4/FoxA binds promoters and recruits RNA polymerase II (Pol II), often in a poised configuration in which Pol II accumulates near transcription start sites. At a later developmental stage, PHA-4 promotes chromatin opening. We found many more genes with poised RNA polymerase than had been observed previously in unstaged embryos, revealing that early embryos accumulate poised Pol II and that poising is dynamic. Our results suggest that Pol II recruitment, in addition to chromatin opening, is an important feature of PHA-4 pioneer factor activity.

Blood-borne hormones acting in the mediobasal hypothalamus, like those controlling food intake, require relatively direct access to target chemosensory neurons of the arcuate nucleus (ARC). An anatomical substrate for this is a permeable microvasculature with fenestrated endothelial cells in the ARC, a system that has awaited comprehensive documentation. Here, the immunofluorescent detection of endothelial fenestral diaphragms in the rat ARC allowed us to quantitate permeable microvessels throughout its rostrocaudal extent. We have determined that permeable microvessels are part of the subependymal plexus irrigating exclusively the ventromedial (vm) ARC from the subadjacent neuroendocrine median eminence. Unexpectedly, permeable microvessels were concentrated proximal to the pituitary stalk. This marked topography strongly supports the functional importance of retrograde blood flow from the pituitary to the vmARC, therefore making a functional relationship between peripheral long-loop, pituitary short-loop, and neuroendocrine ultra-short loop feedback, altogether converging for integration in the vmARC (formerly known as the hypophysiotrophic area), thereby so pivotal as a multicompetent brain endocrinostat.

Estrogen, acting via estrogen receptor (ER)α, regulates serum gonadotropin levels and pituitary gonadotropin subunit expression. However, the cellular pathways mediating this regulation are unknown. ERα signals through classical estrogen response element (ERE)-dependent genomic as well as nonclassical ERE-independent genomic and nongenomic pathways. Using targeted mutagenesis in mice to disrupt ERα DNA binding activity, we previously demonstrated that ERE-independent signaling is sufficient to suppress serum LH levels. In this study, we examined the relative roles of ERE-dependent and -independent estrogen signaling in estrogen regulation of LH, FSH, prolactin, and activin/inhibin subunit gene expression, pituitary LH and FSH protein content, and serum FSH levels. ERE-independent signaling was not sufficient for estrogen to induce pituitary prolactin mRNA or suppress pituitary LHβ mRNA, LH content, or serum FSH in estrogen-treated ovariectomized mice. However, ERE-independent signaling was sufficient to reduce pituitary glycoprotein hormone α-subunit, FSHβ, and activin-βB mRNA expression. Together with previous serum LH results, these findings suggest ERE-independent ERα signaling suppresses serum LH via reduced secretion, not synthesis. Additionally, ERE-dependent and ERE-independent ERα pathways may distinctly regulate steps involved in the synthesis and secretion of FSH.

While the origins of polycystic ovary syndrome (PCOS) in humans are still debated, animal models reliably implicate a fetal origin. Androgen excess, one of the key diagnostic criteria for PCOS and one of its most reliably inherited traits, programs reproductive, adrenal and metabolic organs and tissues during fetal development, producing adult pathology that closely mimics PCOS. Differential gestational timing of androgen excess may also account for heterogeneity in the adult PCOS phenotype. Our fetal or developmental origins hypothesis predicts that development of therapeutic interventions designed to circumvent fetal programming by gestational androgen excess could well eliminate adult PCOS phenotypes.

Although there is extensive evidence that steroids can exert rapid, nongenomic (i.e. nonclassical) actions initiated at the cell surface by binding to specific membrane receptors, the identities of most steroid membrane receptors remain unclear. A novel membrane progestin receptor (mPR) family with seven-transmembrane domains, unrelated to nuclear steroid receptors and comprising three subtypes, alpha, beta and gamma, was recently discovered in fish and other vertebrates. The mPRs are expressed in a wide variety of reproductive tissues. Both recombinant and wild type mPRs display high affinity, saturable, displaceable and specific progestin binding, typical of steroid membrane receptors. Binding and immunoprecipitation studies with [35 S]GTPgam-maS and G-protein alpha subunit antibodies, respectively, show that mPRs are directly coupled to G-proteins and activate them upon progestin treatment. The mPRs typically activate pertussis-sensitive inhibitory G proteins (Gi), to down regulate adenylyl cyclase activity. Recent studies suggest mPRs have important roles in a variety of reproductive functions. Clear evidence has been obtained that mPRs are intermediaries in progestin induction of oocyte maturation in fish and amphibians. The receptor is also involved in progestin stimulation of fish sperm motility. In mammals, the mPRs have been implicated in progesterone regulation of uterine function in humans and GnRH secretion in rodents. The alpha and beta mPR subtypes and their intracellular signaling pathways through Gi have been identified in human myometrial cells. Activation of mPRs in the myometrial cells leads to transactivation of PR-B, the first evidence for cross-talk between membrane and nuclear PRs. Progesterone activation of the mPRs can also lead to a decrease in the expression of the nuclear steroid receptor coactivator SRC2. The data indicate the presence of an unusual signaling pathway mediated by mPRs in human myometria that may result in a functional progestin withdrawal, shifting the balance from a quiescent state to one of contraction at term. Both mPR subtypes are also present in the preoptic anterior hypothalamic region of the rodent brain and in immortalized GnRH-secreting neurons, GT1-7 cells. The finding that rapid down regulation of luteinizing hormone secretion by progesterone also occurs in PRKO mice indicates it is mediated by a novel progesterone receptor. Activation of G proteins and down regulation of adenylyl cyclase activity and GnRH release by GT1-7 cells was observed after progesterone treatment, which was abrogated after transient transfection with siRNA for mPR alpha. The results suggest progesterone decreases GnRH secretion through a mPR/Gi/cAMP pathway.

The ATP-sensitive potassium (KATP) channels are gated by intracellular adenine nucleotides coupling cell metabolism to membrane potential. Channels comprised of Kir6.2 and SUR1 subunits function in subpopulations of mediobasal hypothalamic (MBH) neurons as an essential component of a glucose-sensing mechanism in these cells, wherein uptake and metabolism of glucose leads to increase in intracellular ATP/ADP, closure of the channels, and increase in neuronal excitability. However, it is unknown whether glucose and/or insulin may also regulate the gene expression of the channel subunits in the brain. The present study investigated whether regulation of KATP channel subunit gene expression might be a mechanism by which neuronal populations adapt to prolonged changes in glucose and/or insulin levels in the periphery. Ovariectomized, steroid-replaced rats were fitted with indwelling jugular catheters and infused for 48 h with saline, glucose (hyperglycemia-hyperinsulinemia), insulin and glucose (hyperinsulinemia), diazoxide (control), or glucose and diazoxide (hyperglycemia). At the end of infusions, the MBH, preoptic area, and pituitary were dissected for RNA isolation and RT-PCR. Hyperglycemia decreased Kir6.2 mRNA levels in the MBH in both the presence and absence of hyperinsulinemia. These same conditions also produced a trend toward decreased SUR1 mRNA levels in the MBH; however, it did not exceed statistical significance. Hyperglycemia increased whereas hyperinsulinemia reduced neuropeptide Y mRNA levels when these groups were compared with each other. However, neither was significantly different from values observed in saline-infused controls. In conclusion, hyperglycemia per se may alter expression of KATP channels and thereby induce changes in the excitability of some MBH neurons.

Ovarian estrogen exerts both positive and negative feedback control over luteinizing hormone (LH) secretion during the ovulatory cycle. Estrogen receptor (ER) α but not ERβ knockout mice lack estrogen feedback. Thus, estrogen feedback appears to be primarily mediated by ERα. However, it is now recognized that, in addition to binding to estrogen response elements (EREs) in DNA to alter target gene transcription, ERα signals through ERE-independent or nonclassical pathways, and the relative contributions of these pathways in conveying estrogen feedback remain unknown. Previously we created a knockin mouse model expressing a mutant form of ERα (AA) with ablated ERE-dependent but intact ERE-independent activity. Breeding this allele onto the ERα-null (−/−) background, we examine the ability of ERE-independent ERα signaling pathways to convey estrogen feedback regulation of the female hypothalamic–pituitary axis in vivo . ERα −/AA exhibited 69.9% lower serum LH levels compared with ERα −/− mice. Additionally, like wild type, ERα −/AA mice exhibited elevated LH after ovariectomy (OVX). Furthermore, the post-OVX rise in serum LH was significantly suppressed by estrogen treatment in OVX ERα −/AA mice. However, unlike wild type, both ERα −/AA and ERα −/− mice failed to exhibit estrous cyclicity, spontaneous ovulation, or an afternoon LH surge response to estrogen. These results indicate that ERE-independent ERα signaling is sufficient to convey a major portion of estrogen's negative feedback actions, whereas positive feedback and spontaneous ovulatory cyclicity require ERE-dependent ERα signaling.