Loss-of-Function Mutations in Three Homoeologous PHYTOCLOCK 1 Genes in Common Wheat Are Associated with the Extra-Early Flowering Phenotype.

Triticum aestivum L. cv 'Chogokuwase' is an extra-early flowering common wheat cultivar that is insensitive to photoperiod conferred by the photoperiod insensitive alleles at the Photoperiod-B1 (Ppd-B1) and Ppd-D1loci, and does not require vernalization for flowering. This reduced vernalization requirement is likely due to the spring habitat allele Vrn-D1 at the VERNALIZATION-D1 locus. Genotypes of the Ppd-1 loci that determine photoperiod sensitivity do not fully explain the insensitivity to photoperiod seen in 'Chogokuwase'. We detected altered expression patterns of clock and clock-output genes including Ppd-1 in 'Chogokuwase' that were similar to those in an einkorn wheat mutant that lacks the clock-gene homologue, wheat PHYTOCLOCK 1 (WPCL1). Presumptive loss-of-function mutations in all WPCL1 homoeologous genes were found in 'Chogokuwase' and 'Geurumil', one of the parental cultivars. Segregation analysis of the two intervarietal F2 populations revealed that all the examined F2 plants that headed as early as 'Chogokuwase' had the loss-of-function wpcl1 alleles at all three homoeoloci. Some F2 plants carrying the wpcl1 alleles at three homoeoloci headed later than 'Chogokuwase', suggesting the presence of other loci influencing heading date. Flowering repressor Vrn-2 was up-regulated in 'Chogokuwase' and 'Geurumil' that had the triple recessive wpcl1 alleles. An elevated transcript abundance of Vrn-2 could explain the observation that 'Geurumil' and some F2 plants carrying the three recessive wpcl1 homeoealleles headed later than 'Chogokuwase'. In spite of the up-regulation of Vrn-2, 'Chogokuwase' may have headed earlier due to unidentified earliness genes. Our observations indicated that loss-of-function mutations in the clock gene wpcl1 are necessary but are not sufficient to explain the extra-early heading of 'Chogokuwase'.

A chromatin-dependent role of the fragile X mental retardation protein FMRP in the DNA damage response.

Fragile X syndrome, a common form of inherited intellectual disability, is caused by loss of the fragile X mental retardation protein FMRP. FMRP is present predominantly in the cytoplasm, where it regulates translation of proteins that are important for synaptic function. We identify FMRP as a chromatin-binding protein that functions in the DNA damage response (DDR). Specifically, we show that FMRP binds chromatin through its tandem Tudor (Agenet) domain in vitro and associates with chromatin in vivo. We also demonstrate that FMRP participates in the DDR in a chromatin-binding-dependent manner. The DDR machinery is known to play important roles in developmental processes such as gametogenesis. We show that FMRP occupies meiotic chromosomes and regulates the dynamics of the DDR machinery during mouse spermatogenesis. These findings suggest that nuclear FMRP regulates genomic stability at the chromatin interface and may impact gametogenesis and some developmental aspects of fragile X syndrome.

Proposing a new RNA quadruplex structure: j-motif, with possible links to neural development.

An RNA-binding protein, hnRNP K, has been studied extensively because of its involvement in neural development through the post-transcriptional regulation of its downstream target genes; however, its binding mode remains unclear. According to structural features of the binding sites, we have presumed the existence of possible unique structures 'j-motifs' that are similar to known i-motifs, the difference being that the initial cluster comprises successive U nucleic acids instead of C. It was suspected that the motifs could be recognized by hnRNP K to regulate the translation levels of target proteins, however, there were virtually no methods to verify their existence except computational methods: regular expression searches and theoretical molecular orbital (MO) calculations. Here, we first show a list of 16 genes having j-motif-like sequences we discovered under refined search conditions. The list was highly related to neural development from both subjective and objective aspects. Additionally, MO calculations revealed the similarity of non-canonical base pairs found in i- and j-motifs qualitatively, leading to a proposal of the possible existence of the j-motifs. When taken into consideration, it was indicated that the j-motifs could be formed and play some role in the neural development.

Fragile X syndrome due to a missense mutation.

Fragile X syndrome is a common inherited form of intellectual disability and autism spectrum disorder. Most patients exhibit a massive CGG-repeat expansion mutation in the FMR1 gene that silences the locus. In over two decades since the discovery of FMR1, only a single missense mutation (p.(Ile304Asn)) has been reported as causing fragile X syndrome. Here we describe a 16-year-old male presenting with fragile X syndrome but without the repeat expansion mutation. Rather, we find a missense mutation, c.797G>A, that replaces glycine 266 with glutamic acid (p.(Gly266Glu)). The Gly266Glu FMR protein abolished many functional properties of the protein. This patient highlights the diagnostic utility of FMR1 sequencing.

Reversal of disease-related pathologies in the fragile X mouse model by selective activation of GABAB receptors with arbaclofen.

Fragile X syndrome (FXS), the most common inherited cause of intellectual disability and autism, results from the transcriptional silencing of FMR1 and loss of the mRNA translational repressor protein fragile X mental retardation protein (FMRP). Patients with FXS exhibit changes in neuronal dendritic spine morphology, a pathology associated with altered synaptic function. Studies in the mouse model of fragile X have shown that loss of FMRP causes excessive synaptic protein synthesis, which results in synaptic dysfunction and altered spine morphology. We tested whether the pharmacologic activation of the γ-aminobutyric acid type B (GABA(B)) receptor could correct or reverse these phenotypes in Fmr1-knockout mice. Basal protein synthesis, which is elevated in the hippocampus of Fmr1-knockout mice, was corrected by the in vitro application of the selective GABA(B) receptor agonist STX209 (arbaclofen, R-baclofen). STX209 also reduced to wild-type values the elevated AMPA receptor internalization in Fmr1-knockout cultured neurons, a known functional consequence of increased protein synthesis. Acute administration of STX209 in vivo, at doses that modify behavior, decreased mRNA translation in the cortex of Fmr1-knockout mice. Finally, the chronic administration of STX209 in juvenile mice corrected the increased spine density in Fmr1-knockout mice without affecting spine density in wild-type mice. Thus, activation of the GABA(B) receptor with STX209 corrected synaptic abnormalities considered central to fragile X pathophysiology, a finding that suggests that STX209 may be a potentially effective therapy to treat the core symptoms of FXS.

Central injection of ketone body suppresses luteinizing hormone release via the catecholaminergic pathway in female rats.

Ketosis is found in various pathophysiological conditions, including diabetes and starvation, that are accompanied by suppression of gonadal activity. The aim of the present study was to determine the role of ketone body in the brain in regulating pulsatile luteinizing hormone (LH) secretion in female rats. Injection of 3-hydroxybutyrate (3HB), a ketone body, into the fourth cerebroventricle (4V) induced suppression of pulsatile LH secretion in a dose-dependent manner in ovariectomized (OVX) rats with an estradiol (E2) implant producing diestrus plasma E2 levels. Plasma glucose and corticosterone levels increased immediately after the 4V 3HB injection, suggesting that the treatment caused a hunger response. The 3HB-induced suppression of LH pulses might be mediated by noradrenergic inputs to the hypothalamic paraventricular nucleus (PVN) because a local injection of α-methyl- p-tyrosine, a catecholamine synthesis inhibitor, into the PVN blocked 3HB-induced suppression of LH pulses and PVN noradrenaline release was increased by 4V 3HB injection in E2-primed OVX rats. These results suggest that ketone body sensed by a central energy sensor in the hindbrain may suppress gonadotropin release via noradrenergic inputs to the PVN under ketosis.

Involvement of brain ketone bodies and the noradrenergic pathway in diabetic hyperphagia in rats.

Uncontrolled type 1 diabetes leads to hyperphagia and severe ketosis. This study was conducted to test the hypothesis that ketone bodies act on the hindbrain as a starvation signal to induce diabetic hyperphagia. Injection of an inhibitor of monocarboxylate transporter 1, a ketone body transporter, into the fourth ventricle normalized the increase in food intake in streptozotocin (STZ)-induced diabetic rats. Blockade of catecholamine synthesis in the hypothalamic paraventricular nucleus (PVN) also restored food intake to normal levels in diabetic animals. On the other hand, hindbrain injection of the ketone body induced feeding, hyperglycemia, and fatty acid mobilization via increased sympathetic activity and also norepinephrine release in the PVN. This result provides evidence that hyperphagia in STZ-induced type 1 diabetes is signaled by a ketone body sensed in the hindbrain, and mediated by noradrenergic inputs to the PVN.

Significance of neonatal testicular sex steroids to defeminize anteroventral periventricular kisspeptin neurons and the GnRH/LH surge system in male rats.

The brain mechanism regulating gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release is sexually differentiated in rodents. Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) have been suggested to be sexually dimorphic and involved in the GnRH/LH surge generation. The present study aimed to determine the significance of neonatal testicular androgen to defeminize AVPV kisspeptin expression and the GnRH/LH surge-generating system. To this end, we tested whether neonatal castration feminizes AVPV kisspeptin neurons and the LH surge-generating system in male rats and whether neonatal estradiol benzoate (EB) treatment suppresses the kisspeptin expression and the LH surge in female rats. Immunohistochemistry, in situ hybridization, and quantitative real-time RT-PCR were performed to investigate kisspeptin and Kiss1 mRNA expressions. Male rats were castrated immediately after birth, and females were treated with EB on postnatal Day 5. Neonatal castration caused an increase in AVPV kisspeptin expression at peptide and mRNA levels in the genetically male rats, and the animals showed surge-like LH release in the presence of the preovulatory level of estradiol (E2) at adulthood. On the other hand, neonatal EB treatment decreased the number of AVPV kisspeptin neurons and caused an absence of E2-induced LH surge in female rats. Semiquantitative RT-PCR analysis showed that neonatal steroidal manipulation affects Kiss1 expression but does not significantly affect gene expressions of neuropeptides (neurotensin and galanin) and enzymes or transporter for neurotransmitters (gamma-aminobutyric acid, glutamate, and dopamine) in the AVPV, suggesting that the manipulation specifically affects Kiss1 expressions. Taken together, our present results provide physiological evidence that neonatal testicular androgen causes the reduction of AVPV kisspeptin expression and failure of LH surge in genetically male rats. Thus, it is plausible that perinatal testicular androgen causes defeminization of the AVPV kisspeptin system, resulting in the loss of the surge system in male rats.

Paraventricular alpha1- and alpha2-adrenergic receptors mediate hindbrain lipoprivation-induced suppression of luteinizing hormone pulses in female rats.

Acute central lipoprivation suppresses pulsatile luteinizing hormone (LH) release and increases blood glucose levels through noradrenergic input to the hypothalamic paraventricular nucleus (PVN) in female rats. The present study was conducted to identify adrenergic receptor subtypes involved in central lipoprivation-induced suppression of pulsatile LH secretion and increases in plasma glucose levels in female rats. Acute hindbrain lipoprivation was produced by injection into the fourth cerebroventricle (4V) of 2-mercaptoacetate (MA), an inhibitor of fatty acid oxidation, in estradiol-implanted ovariectomized rats. Two min before MA injection, alpha1-, alpha2- or beta-adrenergic receptor antagonist was injected into the PVN. Injection of MA into the 4V suppresses pulsatile LH release in PVN vehicle-treated rats, whereas pretreatment of animals with injection of alpha1- or alpha2-adrenergic antagonist into the PVN blocked the effect of the 4V MA injection on LH pulses. beta-Adrenergic antagonist did not affect MA-induced suppression of LH pulses. The counter-regulatory increase in plasma glucose levels after 4V MA injection was also partially blocked by pretreatment with alpha1- and alpha2-adrenergic receptor antagonists. These results suggest that alpha1- and alpha2-adrenergic receptors in the PVN mediate hindbrain lipoprivation-induced suppression of LH release and counter-regulatory increases in plasma glucose levels in female rats.

Central lipoprivation-induced suppression of luteinizing hormone pulses is mediated by paraventricular catecholaminergic inputs in female rats.

The present study aims to clarify the role of fatty acids in regulating pulsatile LH secretion in rats. To produce an acute central lipoprivic condition, mercaptoacetate (MA), an inhibitor of fatty acids oxidation, was administered into the fourth cerebroventricle (4V) in ad libitum fed ovariectomized (OVX) rats (0.4, 2, and 10 micromol/rat) with or without an estradiol (E2) implant producing diestrus plasma E2 levels. Pulsatile LH secretion was suppressed by 4V MA administration in a dose-dependent manner in both OVX and OVX plus E2 rats. Mean LH levels and LH pulse frequency and amplitude were significantly reduced by the highest dose of MA in OVX rats, and by the middle and highest dose of MA in E2-treated rats, suggesting that estrogen enhanced LH suppression. Blood glucose levels increased immediately after the highest dose of MA in both groups. Fourth ventricular injection of trimetazidine (2 and 3 micromol/rat), another inhibitor of fatty acids oxidation, also inhibited pulsatile LH release, resulting in significant and dose-dependent suppression of LH pulse frequency and an increase in blood glucose levels in OVX plus E2 rats. In contrast, peripheral injection of the highest 4V dose of MA (10 micromol/rat) did not alter LH release or blood glucose levels. Microdialysis of the hypothalamic paraventricular nucleus (PVN) revealed that norepinephrine release in the region was increased by 4V MA administration. Preinjection of alpha-methyl-p-tyrosine, a catecholamine synthesis inhibitor, into the PVN completely blocked the lipoprivic inhibition of LH and the counter-regulatory increase in blood glucose levels in OVX plus E2 rats. Together, these studies indicate that fatty acid availability may be sensed by a central detector, located in the lower brainstem to maintain reproduction, and that noradrenergic inputs to the PVN mediate this lipoprivic-induced suppression of LH release.

Involvement of anteroventral periventricular metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats.

Metastin/kisspeptin, the KiSS-1 gene product, has been identified as an endogenous ligand of GPR54 that reportedly regulates GnRH/LH surges and estrous cyclicity in female rats. The aim of the present study was to determine if metastin/kisspeptin neurons are a target of estrogen positive feedback to induce GnRH/LH surges. We demonstrated that preoptic area (POA) infusion of the anti-rat metastin/kisspeptin monoclonal antibody blocked the estrogen-induced LH surge, indicating that endogenous metastin/kisspeptin released around the POA mediates the estrogen positive feedback effect on GnRH/LH release. Metastin/kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) may be responsible for mediating the feedback effect because the percentage of c-Fos-expressing KiSS-1 mRNA-positive cells to total KiSS-1 mRNA-positive cells was significantly higher in the afternoon than in the morning in the anteroventral periventricular nucleus (AVPV) of high estradiol (E(2))-treated females. The percentage of c-Fos-expressing metastin/kisspeptin neurons was not different between the afternoon and morning in the arcuate nucleus (ARC). Most of the KiSS-1 mRNA expressing cells contain ERalpha immunoreactivity in the AVPV and ARC. In addition, AVPV KiSS-1 mRNA expressions were highest in the proestrous afternoon and lowest in the diestrus 1 in females and were increased by estrogen treatment in ovariectomized animals. On the other hand, the ARC KiSS-1 mRNA expressions were highest at diestrus 2 and lowest at proestrous afternoon and were increased by ovariectomy and decreased by high estrogen treatment. Males lacking the surge mode of GnRH/LH release showed no obvious cluster of metastin/kisspeptin-immunoreactive neurons in the AVPV when compared with high E(2)-treated females, which showed a much greater density of these neurons. Taken together, the present study demonstrates that the AVPV metastin/kisspeptin neurons are a target of estrogen positive feedback to induce GnRH/LH surges in female rats.

Acute lipoprivation suppresses pulsatile luteinizing hormone secretion without affecting food intake in female rats.

The present study examined the effect of acute lipoprivation on pulsatile luteinizing hormone (LH) secretion in both normal-fat diet, ad libitum-fed and fasted female rats. To produce an acute lipoprivic condition, mercaptoacetate (MA), an inhibitor of fatty acid oxidation, was administered intraperitoneally to ad libitum-fed or 24-h fasted ovariectomized (OVX) rats with or without an estradiol (E2) implant, that produces a negative feedback effect on LH pulses. The steroid treatment was performed to determine the effect of estrogen on lipoprivic changes in LH release, because estrogen enhances fasting- or glucoprivation-induced suppression of LH pulses. Pulsatile LH secretion was suppressed by MA administration in a dose-dependent manner in the ad libitum-fed OVX and OVX+E2 rats. LH pulses were more severely suppressed in the 24-h-fasted OVX and OVX+E2 rats compared to the ad libitum-fed rats. Estrogen slightly enhanced lipoprivic suppression but the effect was not significant. In the present study, increased plasma glucose and free-fatty acid concentrations may indicate a blockade of fatty acid metabolism by the MA treatment, but food intake was not affected by any of the MA doses. Acute vagotomy did not block lipoprivic suppression of LH pulses. Thus, the present study indicates that lipid metabolism is important for maintenance of normal reproductive function even in rats fed a normal-fat diet and lipoprivation may be more critical in fasted animals that probably rely more heavily on fatty acid oxidation to maintain appropriate metabolic fuel levels. In addition, failure of blockade of lipoprivic LH inhibition by vagotomy suggests that lipoprivic information resulting in LH suppression is not transmitted to the brain via the vagus nerve.

Involvement of central metastin in the regulation of preovulatory luteinizing hormone surge and estrous cyclicity in female rats.

Ovulation is caused by a sequence of neuroendocrine events: GnRH and LH surges that are induced by positive feedback action of estrogen secreted by the mature ovarian follicles. The central mechanism of positive feedback action of estrogen on GnRH/LH secretion, however, is not fully understood yet. The present study examined whether metastin, the product of metastasis suppressor gene KiSS-1, is a central neuropeptide regulating GnRH/LH surge and then estrous cyclicity in the female rat. Metastin had a profound stimulation on LH secretion by acting on the preoptic area (POA), where most GnRH neurons projecting to the median eminence are located, because injection of metastin into the third ventricle or POA increased plasma LH concentrations in estrogen-primed ovariectomized rats. Metastin neurons were immunohistochemically found in the arcuate nucleus (ARC) to be colocalized with estrogen receptors with some fibers in the preoptic area (POA) in close apposition with GnRH neuronal cell bodies or fibers. Quantitative RT-PCR has revealed that KiSS-1 and GPR54 mRNAs were expressed in the ARC and POA, respectively. The blockade of local metastin action in the POA with a specific monoclonal antibody to rat metastin completely abolished proestrous LH surge and inhibited estrous cyclicity. Metastin-immunoreactive cell bodies in the ARC showed a marked increase and c-Fos expression in the early proestrus afternoon compared with the day of diestrus. Thus, metastin released in the POA is involved in inducing the preovulatory LH surge and regulating estrous cyclicity.

Fourth ventricular alloxan injection suppresses pulsatile luteinizing hormone release in female rats.

Previous studies have suggested the presence of a glucose-sensing mechanism in the hindbrain that appears to regulate reproductive function as well as feeding behavior. The ependymocytes lining the ventricular wall of the hindbrain showed immunoreactivities to pancreatic glucokinase (GK), a key enzyme for glucose sensing in pancreatic B cells. Our goal in the present study was to test whether the GK-immunopositive ependymocytes in the wall of the fourth cerebroventricle (4V) play a role in regulating gonadal activity. Our approach was to determine the effect of injecting alloxan, a GK inhibitor, into the 4V on pulsatile luteinizing hormone (LH) secretion. Estrogen-primed ovariectomized rats received an injection of alloxan (10 or 20 microg/animal) into the 4V and blood samples were collected every 6 min for 3 h for measurement of blood LH, corticosterone and glucose levels. Pulsatile LH secretion was suppressed after alloxan injection and all pulse parameters were significantly (P<0.05) inhibited by 20 microg alloxan. Plasma corticosterone levels were increased significantly (P<0.05) by 20 microg alloxan, suggesting that LH pulse suppression by alloxan may be at least partly mediated by activation of the hypothalamo-pituitary-adrenal axis. The present results suggest that acute suppression of GK activity in the hindbrain inhibits pulsatile LH secretion in female rats, and supports the idea that GK-immunopositive ependymocytes may sense glucose levels in the cerebrospinal fluid and play a role in regulation of LH secretion.

In vitro increase in intracellular calcium concentrations induced by low or high extracellular glucose levels in ependymocytes and serotonergic neurons of the rat lower brainstem.

Pancreatic glucokinase (GK)-like immunoreactivities are located in ependymocytes and serotonergic neurons of the rat brain. The present study investigated in vitro changes in intracellular calcium concentrations ([Ca(2+)](i)) in response to low (2 mm) or high (20 mm) extracellular glucose concentrations in isolated cells from the wall of the central canal (CC), raphe obscurus nucleus (ROb), ventromedial hypothalamus (VMH), and lateral hypothalamic area (LHA) in male rats. An increase in [Ca(2+)](i) was found in cells from the CC (21.1% or 9.8% of ependymocytes), ROb (10.9% or 14.5% of serotonergic neurons), VMH (7.8% and 25.2% of neurons), and LHA (20% or 15.7% of neurons), when extracellular glucose levels were changed from 10 to either 2 or 20 mm, respectively. Most of the ependymocytes and serotonergic neurons responding to the glucose changes were immunoreactive to the anti-GK in the CC (96.8% for low glucose and 100% for high glucose) and ROb (100% for low and high glucose). The [Ca(2+)](i) increase was blocked with calcium-free medium or L-type calcium channel blocker. Cells with an increase in [Ca(2+)](i) in response to low glucose did not respond to high glucose and vice versa. Inhibition of GK activity with acute alloxan treatment blocked low or high glucose-induced [Ca(2+)](i) increases in most GK-immunoreactive cells from the CC or ROb. The glucose-sensitive [Ca(2+)](i) increase in neurons of the VMH and LHA was also alloxan-sensitive, but no cells taken from the VMH and LHA were immunoreactive to the antibody used. The present study further indicates that ependymocytes of the CC and serotonergic neurons in the ROb are also sensitive to the changes in extracellular glucose in a GK-dependent manner, but that the subtype of GK in these cells could be different from that in the VMH and LHA.

Norepinephrine triggers Ca2+-dependent exocytosis of 5-hydroxytryptamine from rat pinealocytes in culture.

5-hydroxytryptamine (5-HT) is a precursor and a putative modulator for melatonin synthesis in mammalian pinealocytes. 5-HT is present in organelles distinct from l-glutamate-containing synaptic-like microvesicles as well as in the cytoplasm of pinealocytes, and is secreted upon stimulation by norepinephrine (NE) to enhance serotonin N-acetyltransferase activity via the 5-HT2 receptor. However, the mechanism underlying the secretion of 5-HT from pinealocytes is unknown. In this study, we show that NE-evoked release of 5-HT is largely dependent on Ca2+ in rat pinealocytes in culture. Omission of Ca2+ from the medium and incubation of pineal cells with EGTA-tetraacetoxymethyl-ester inhibited by 59 and 97% the NE-evoked 5-HT release, respectively. Phenylephrine also triggered the Ca2+-dependent release of 5-HT, which was blocked by phentolamine, an alpha antagonist, but not by propranolol, a beta antagonist. Botulinum neurotoxin type E cleaved 25 kDa synaptosomal-associated protein and inhibited by 50% of the NE-evoked 5-HT release. Bafilomycin A1, an inhibitor of vacuolar H+-ATPase, and reserpine and tetrabenazine, inhibitors of vesicular monoamine transporter, all decreased the storage of vesicular 5-HT followed by inhibition of the NE-evoked 5-HT release. Agents that trigger L-glutamte exocytosis such as acetylcholine did not trigger any Ca2+-dependent 5-HT release. Vice versa neither NE nor phenylephrine caused synaptic-like microvesicle-mediated l-glutamate exocytosis. These results indicated that upon stimulation of a adrenoceptors pinealocytes secrete 5-HT through a Ca2+-dependent exocytotic mechanism, which is distinct from the exocytosis of synaptic-like microvesicles.