Aralkylamine N-acetyltransferase Research Articles

Role of Aralkylamine N-Acetyltransferase in the Response to Antioxidative Stress in the Fruit Fly Drosophila Melanogaster Adults.

In multicellular organisms, the indole melatonin synthesized by aralkylamine N-acetyltransferase (AANATI) serves as an antioxidant. To test this, sex-mixed 3-day-old mated fly adults bw1 and AANAT1 homozygous recessive loss-of-function mutant (bw AANAT1lo) of Drosophila melanogaster were fed by a standard diet or by one containing paraquat (PQ, 1,1'-dimethyl-4,4'-bipyridilium dichloride hydrate) at a final concentration of 15.5 mM. Experiment lasted 8 h and began at 11 a.m. In bw1 flies the paraquat treatment resulted in a significant (evaluated by Student's t-tests) decrease of the superoxide dismutase (SOD) activity and an increase the catalase (CAT) and glutathione S-transferase (GST) activities. Meanwhile, in these flies, total Antioxidative capacity (TAC) was significantly curbed by the paraquat presence. Importantly, these changes were not observed in the AANAT1-mutants. Thus, melatonin seems to play an important defence role against the oxidative stress elicited by paraquat.

Biosynthesis of neuroprotective melatonin is dysregulated in Huntington's disease.

Huntington's disease (HD) is a progressive neurodegenerative brain disorder associated with uncontrolled body movements, cognitive decline, and reduced circulating melatonin levels. Melatonin is a potent antioxidant and exogenous melatonin treatment is neuroprotective in experimental HD models. In neurons, melatonin is exclusively synthesized in the mitochondrial matrix. Thus, we investigated the integrity of melatonin biosynthesis pathways in pineal and extrapineal brain areas in human HD brain samples, in the R6/2 mouse model of HD and in full-length mutant huntingtin knock-in cells. Aralkylamine N-acetyltransferase (AANAT) is the rate-limiting step enzyme in the melatonin biosynthetic pathway. We found that AANAT expression is significantly decreased in the pineal gland and the striatum of HD patients compared to normal controls. In the R6/2 mouse forebrain, AANAT protein expression was decreased in synaptosomal, but not nonsynaptosomal, mitochondria and was associated with decreased synaptosomal melatonin levels compared to wild type mice. We also demonstrate sequestration of AANAT in mutant-huntingtin protein aggregates likely resulting in decreased AANAT bioavailability. Paradoxically, AANAT mRNA expression is increased in tissues where AANAT protein expression is decreased, suggesting a potential feedback loop that is, ultimately unsuccessful. In conclusion, we demonstrate that pineal, extrapineal, and synaptosomal melatonin levels are compromised in the brains of HD patients and R6/2 mice due, at least in part, to protein aggregation.

The ISL LIM-homeobox 2 transcription factor is negatively regulated by circadian adrenergic signaling to repress the expression of Aanat in pinealocytes of the rat pineal gland.

Melatonin is synthesized in the pineal gland during nighttime in response to nocturnal increase in the activity of the enzyme aralkylamine N-acetyltransferase (AANAT), the transcription of which is modulated by several homeodomain transcription factors. Recent work suggests that the homeodomain transcription factor ISL LIM homeobox 2 (ISL2) is expressed in the pineal gland, but its role is currently unknown. With the purpose of identifying the mechanisms that control pineal expression of Isl2 and the possible function of Isl2 in circadian pineal biology, we report that Isl2 is specifically expressed in the pinealocytes of the rat pineal gland. Its expression exhibits a 24 h rhythm with high transcript and protein levels during the day and a trough in the second half of the night. This rhythm persists in darkness, and lesion studies reveal that it requires intact function of the suprachiasmatic nuclei, suggesting intrinsic circadian regulation. In vivo and in vitro experiments show that pineal Isl2 expression is repressed by adrenergic signaling acting via cyclic AMP; further, Isl2 is negatively regulated by the nocturnal transcription factor cone-rod homeobox. During development, pineal Isl2 expression is detectable from embryonic day 19, preceding Aanat by several days. In vitro knockdown of Isl2 is accompanied by an increase in Aanat transcript levels suggesting that ISL2 represses its daytime expression. Thus, rhythmic expression of ISL2 in pinealocytes is under the control of the suprachiasmatic nucleus acting via adrenergic signaling in the gland to repress nocturnal expression, while ISL2 itself negatively regulates daytime pineal expression of Aanat and thereby suggestively enhances the circadian rhythm in melatonin synthesis.

Methadone administered to rat dams during pregnancy and lactation affects the circadian rhythms of their pups.

The circadian clock is one of the most important homeostatic systems regulating the majority of physiological functions. Its proper development contributes significantly to the maintenance of health in adulthood. Methadone is recommended for the treatment of opioid use disorders during pregnancy, increasing the number of children prenatally exposed to long-acting opioids. Although early-life opioid exposure has been studied for a number of behavioral and physiological changes observed later in life, information on the relationship between the effects of methadone exposure and circadian system development is lacking. Using a rat model, we investigated the effects of prenatal and early postnatal methadone administration on the maturation of the circadian clockwork in the suprachiasmatic nucleus (SCN) and liver, the rhythm of aralkylamine N-acetyltransferase (AA-NAT) activity in the pineal gland, and gene expression in the livers of 20-day-old rats. Our data show that repeated administration of methadone to pregnant and lactating mothers has significant effect on rhythmic gene expression in the SCN and livers and on the rhythm of AA-NAT in the offspring. Similar to previous studies with morphine, the rhythm amplitudes of the clock genes in the SCN and liver were unchanged or enhanced. However, six of seven specific genes in the liver showed significant downregulation of their expression, compared to the controls in at least one experimental group. Importantly, the amplitude of the AA-NAT rhythm was significantly reduced in all methadone-treated groups. As there is a strong correlation with melatonin levels, this result could be of importance for clinical practice.

The Photoperiod-Driven Cyclical Secretion of Pineal Melatonin Regulates Seasonal Reproduction in Geese (Anser cygnoides).

The photoperiod is the predominant environmental factor that governs seasonal reproduction in animals; however, the underlying molecular regulatory mechanism has yet to be fully elucidated. Herein, Yangzhou geese (Anser cygnoides) were selected at the spring equinox (SE), summer solstice (SS), autumn equinox (AE), and winter solstice (WS), and the regulation of seasonal reproduction via the light-driven cyclical secretion of pineal melatonin was investigated. We show that there were seasonal variations in the laying rate and GSI, while the ovarian area decreased 1.5-fold from the SS to the AE. Moreover, not only did the weight and volume of the pineal gland increase with a shortened photoperiod, but the secretory activity was also enhanced. Notably, tissue distribution further revealed seasonal oscillations in melatonin receptors (Mtnrs) in the pineal gland and the hypothalamus-pituitary-gonadal (HPG) axis. The immunohistochemical staining indicated higher Mtnr levels due to the shortened photoperiod. Furthermore, the upregulation of aralkylamine N-acetyltransferase (Aanat) was observed from the SS to the AE, concurrently resulting in a downregulation of the gonadotrophin-releasing hormone (GnRH) and gonadotropins (GtHs). This trend was also evident in the secretion of hormones. These data indicate that melatonin secretion during specific seasons is indicative of alterations in the photoperiod, thereby allowing for insight into the neuroendocrine regulation of reproduction via an intrinsic molecular depiction of external photoperiodic variations.

The Role of the PAA1 Gene on Melatonin Biosynthesis in Saccharomyces cerevisiae: A Search of New Arylalkylamine N-Acetyltransferases

Recently, the presence of melatonin in fermented beverages has been correlated with yeast metabolism during alcoholic fermentation. Melatonin, originally considered a unique product of the pineal gland of vertebrates, has been also identified in a wide range of invertebrates, plants, bacteria, and fungi in the last two decades. These findings bring the challenge of studying the function of melatonin in yeasts and the mechanisms underlying its synthesis. However, the necessary information to improve the selection and production of this interesting molecule in fermented beverages is to disclose the genes involved in the metabolic pathway. So far, only one gene has been proposed as involved in melatonin production in Saccharomyces cerevisiae, PAA1, a polyamine acetyltransferase, a homolog of the vertebrate’s aralkylamine N-acetyltransferase (AANAT). In this study, we assessed the in vivo function of PAA1 by evaluating the bioconversion of the different possible substrates, such as 5-methoxytryptamine, tryptamine, and serotonin, using different protein expression platforms. Moreover, we expanded the search for new N-acetyltransferase candidates by combining a global transcriptome analysis and the use of powerful bioinformatic tools to predict similar domains to AANAT in S. cerevisiae. The AANAT activity of the candidate genes was validated by their overexpression in E. coli because, curiously, this system evidenced higher differences than the overexpression in their own host S. cerevisiae. Our results confirm that PAA1 possesses the ability to acetylate different aralkylamines, but AANAT activity does not seem to be the main acetylation activity. Moreover, we also prove that Paa1p is not the only enzyme with this AANAT activity. Our search of new genes detected HPA2 as a new arylalkylamine N-acetyltransferase in S. cerevisiae. This is the first report that clearly proves the involvement of this enzyme in AANAT activity.

The Uterine Melatonergic Systems of AANAT and Melatonin Membrane Receptor 2 (MT2) Are Essential for Endometrial Receptivity and Early Implantation in Mice.

In the current study, using Aanat and Mt2 KO mice, we observed that the preservation of the melatonergic system is essential for successful early pregnancy in mice. We identified that aralkylamine N-acetyltransferase (AANAT), melatonin receptor 1A (MT1), and melatonin receptor 1B (MT2) were all expressed in the uterus. Due to the relatively weak expression of MT1 compared to AANAT and MT2, this study focused on AANAT and MT2. Aanat and Mt2 KO significantly reduced the early implantation sites and the abnormal morphology of the endometrium of the uterus. Mechanistical analysis indicated that the melatonergic system is the key player in the induction of the normal nidatory estrogen (E2) response for endometrial receptivity and functions by activating the STAT signaling pathway. Its deficiency impaired the interactions between the endometrium, the placenta, and the embryo. The reduction in melatonin production caused by Aanat KO and the impairment of signal transduction caused by Mt2 KO reduced the uterine MMP-2 and MMP-9 activity, resulting in a hyperproliferative endometrial epithelium. In addition, melatonergic system deficiency also increased the local immunoinflammatory reaction with elevated local proinflammatory cytokines leading to early abortion in the Mt2 KO mice compared to the WT mice. We believe that the novel data obtained from the mice might apply to other animals including humans. Further investigation into the interaction between the melatonergic system and reproductive effects in different species would be worthwhile.

Artificial light at night triggers slight transcriptomic effects on melatonin signaling but not synthesis in tadpoles of two anuran species

The worldwide expansion of artificial light at night (ALAN) is acknowledged as a threat to biodiversity through alterations of the natural photoperiod triggering the disruption of physiological functions. In vertebrates, melatonin production during the dark phase can be decreased or suppressed by nocturnal light as shown in many taxa. But the effect of ALAN at low intensity mimicking light pollution in peri-urban area has never been investigated in amphibians. We filled this gap by studying the impact of low ALAN levels on the expression of genes related to melatonin synthesis and signaling in two anurans (agile frog, Rana dalmatina, and common toad, Bufo bufo). Circadian expression of genes encoding enzymes catalyzing melatonin synthesis (aralkylamine N-acetyltransferase, AANAT and acetylserotonin O-methyltransferase, ASMT) or melatonin receptors (Mel1a, Mel1b and Mel1c) was investigated using RT-qPCR after 23 days of nocturnal exposure to control (< 0.01 lx) or low ALAN (3 lx). We showed that the relative abundance of most transcripts was low in late afternoon and early evening (06 pm and 08 pm) and increased throughout the night in R. dalmatina. However, a clear and ample nocturnal pattern of target gene expression was not detected in control tadpoles of both species. Surprisingly, a low ALAN level had little influence on the relative expression of most melatonin-related genes. Only Mel1c expression in R. dalmatina and Mel1b expression in B. bufo were affected by ALAN. This target gene approach provides experimental evidence that melatonin signaling pathway was slightly affected by low ALAN level in anuran tadpoles.

Effects of different photoperiod conditions on survival, growth, and gonadal development of Takifugu rubripes adults

Photoperiod has a significant influence on fish. In this study, 2.5-year-old Takifugu rubripes (tiger puffer) adults were selected to evaluate the photoperiod impacts (8 h light (L):16 h dark (D), 12L:12D, 16L:8D) from September to January at 15–17 °C on their growth, gonadal development, and survival for a 100-day. The mortality rate of males and females tiger puffer differed among the different photoperiods, and the 8L:16D photoperiod was not suitable for survival of males. At the end of the experiment, the body length, weight gain rate, specific growth rate and condition factor of both males and females tiger puffer were similar among the three photoperiods (P > 0.05). Higher percentage of females had developed to later developing phase and males had developed to spawning capable phase in the 12L:12D treatment, respectively. In particular, 100% of males had testis at the spawning capable phase. The gonadosomatic index values of males and females in the 12L:12D group were significantly higher than those in the 16L:8D group (P < 0.05), but no significant differences between the 8L:16D and 12L:12D groups (P > 0.05). The serum testosterone (T) content of males and the serum estradiol-17β (E2) content of females were highest in the 12L:12D group. The expression of aralkylamine N-acetyltransferase 1a (aanat1a) in the diencephalon/midbrain (D/M) was elevated under 12L:12D treatment in both sex (P < 0.05). The expression level of iodothyronine deiodinase type 2 (dio2) in the saccus vasculosus in males was elevated under the 12L:12D photoperiod (P < 0.05). The levels of gonadotropin-releasing hormone 1 (gnrh1) and gnrh3 mRNA in the D/M of females at later developing stages in the 12L:12D treatment were significantly higher than those in of other treatment groups, and the level of gnrh3 in males under this photoperiod was significantly elevated (P < 0.05). The levels of gonadotropin-inhibiting hormone (gnih) mRNA under 12L:12D were significantly elevated in both males and females (P < 0.05). Under this photoperiod, the levels of follicle stimulating hormone b-subunit (fshb) and luteinizing hormone b-subunit (lhb) in the pituitary gland of females at later developing stages were significantly higher than those of the other two groups (P < 0.05). The expression levels of lhb in males in the 12L:12D and 8L:16D photoperiods were significantly higher than that at 16L:8D (P < 0.05). These results indicated that culture under the 12L:12D photoperiod from September prior to the spawning season could result in accelerated gonadal development of both male and female tiger puffer.

Effects of SNPs in AANAT and ASMT Genes on Milk and Peripheral Blood Melatonin Concentrations in Holstein Cows (Bos taurus).

Aralkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT), the two rate-limiting enzymes for melatonin synthesis, regulate melatonin production in mammals. Through analysis of the milk melatonin level and dairy herd improvement (DHI) index, it was found that the melatonin concentration in milk was significantly negatively correlated with the 305 day milk yield (305M) and peak milk yield (PeakM) (p < 0.05), while it was significantly positively correlated with the serum melatonin concentration (p < 0.05). The full-length of AANAT and ASMT were sequenced and genotyped in 122 cows. Three SNPs in AANAT and four SNPs in ASMT were significantly related to MT levels in the milk and serum (p < 0.05). The SNPs in AANAT were temporarily denoted as N-SNP1 (g.55290169 T>C), N-SNP2 (g.55289357 T>C), and N-SNP3 (g.55289409 C>T). The SNPs in ASMT were temporarily denoted as M-SNP1 (g.158407305 G>A), M-SNP2 (g.158407477 A>G), M-SNP3 (g.158407874 G>A), and M-SNP4 (g.158415342 T>C). The M-SNP1, M-SNP2, and M-SNP3 conformed to the Hardy−Weinberg equilibrium (p > 0.05), while other SNPs deviated from the Hardy−Weinberg equilibrium (p < 0.05). The potential association of MT production and each SNP was statistically analyzed using the method of linkage disequilibrium (LD). The results showed that N-SNP2 and N-SNP3 had some degree of LD (D′ = 0.27), but M-SNP1 and M-SNP2 had a strong LD (D′ = 0.98). Thus, the DHI index could serve as a prediction of the milk MT level. The SNPs in AANAT and ASMT could be used as potential molecular markers for screening cows to produce high melatonin milk.

Incubation Temperature and Lighting: Effect on Embryonic Development, Post-Hatch Growth, and Adaptive Response.

During incubation, the content of the egg is converted into a chick. This process is controlled by incubation conditions, which must meet the requirements of the chick embryo to obtain the best chick quality and maximum hatchability. Incubation temperature and light are the two main factors influencing embryo development and post-hatch performance. Because chicken embryos are poikilothermic, embryo metabolic development relies on the incubation temperature, which influences the use of egg nutrients and embryo development. Incubation temperature ranging between 37 and 38°C (typically 37.5–37.8°C) optimizes hatchability. However, the temperature inside the egg called “embryo temperature” is not equal to the incubator air temperature. Moreover, embryo temperature is not constant, depending on the balance between embryonic heat production and heat transfer between the eggshell and its environment. Recently, many studies have been conducted on eggshell and/or incubation temperature to meet the needs of the embryo and to understand the embryonic requirements. Numerous studies have also demonstrated that cyclic increases in incubation temperature during the critical period of incubation could induce adaptive responses and increase the thermotolerance of chickens without affecting hatchability. Although the commercial incubation procedure does not have a constant lighting component, light during incubation can modify embryo development, physiology, and post-hatch behavior indicated by lowering stress responses and fearful behavior and improving spatial abilities and cognitive functions of chicken. Light-induced changes may be attributed to hemispheric lateralization and the entrainment of circadian rhythms in the embryo before the hatching. There is also evidence that light affects embryonic melatonin rhythms associated with body temperature regulation. The authors’ preliminary findings suggest that combining light and cyclic higher eggshell temperatures during incubation increases pineal aralkylamine N-acetyltransferase, which is a rate-limiting enzyme for melatonin hormone production. Therefore, combining light and thermal manipulation during the incubation could be a new approach to improve the resistance of broilers to heat stress. This review aims to provide an overview of studies investigating temperature and light manipulations to improve embryonic development, post-hatch growth, and adaptive stress response in chickens.

Electroacupuncture Ameliorates Cognitive Impairment Through the Inhibition of NLRP3 Inflammasome Activation by Regulating Melatonin-Mediated Mitophagy in Stroke Rats.

Previous studies found that electroacupuncture (EA) at the Shenting (DU24) and Baihui (DU20) acupoints alleviates cognitive impairment in cerebral ischemia-reperfusion (I/R) injury rats. Nonetheless, the mechanisms of the anti-inflammatory effects of EA are unclear. Cerebral I/R injury was induced in rats by middle cerebral artery occlusion (MCAO). Following I/R injury, the rats underwent EA therapy at the Shenting (DU24) and Baihui (DU20) acupoints for seven successive days. The Morris water maze test, magnetic resonance imaging (MRI) and molecular biology assays were utilized to assess the establishment of the rat stroke model with cognitive impairment and the therapeutic effect of EA. EA treatment of rats subjected to MCAO showed a significant reduction in infarct volumes accompanied by cognitive recovery, as observed in Morris water maze test outcomes. The possible mechanisms by which EA treatment attenuates cognitive impairment are by regulating endogenous melatonin secretion through aralkylamine N-acetyltransferase gene (AANAT, a rate-limiting enzyme of melatonin) synthesis in the pineal gland in stroke rats. Simultaneously, through melatonin regulation, EA exerts neuroprotective effects by upregulating mitophagy-associated proteins and suppressing reactive oxygen species (ROS)-induced NLRP3 inflammasome activation after I/R injury. However, melatonin receptor inhibitor (luzindole) treatment reversed these changes. The findings from this research suggested that EA ameliorates cognitive impairment through the inhibition of NLRP3 inflammasome activation by regulating melatonin-mediated mitophagy in stroke rats.

Effect of short- and long-term melatonin treatments on the reproductive activity of the tropical damselfish Chrysiptera cyanea.

Photoperiod plays a role in controlling the initiation and termination of reproduction in fish. Melatonin is an internal transducer of environmental photoperiod and is involved in regulating reproduction. The present study aimed to examine how melatonin impacts the transcript levels of kisspeptin (kiss1 and kiss2), gonadotropin-releasing hormones (gnrh1), and the β-subunit of gonadotropins (fshβ and lhβ) in the brain of the sapphire devil, a tropical damselfish with long photoperiod preference. Feeding mature females with melatonin-containing pellets inhibited increases in the transcript levels of kiss1, gnrh1, and lhβ within 3 h. Continuous melatonin treatment for 1 week resulted in oocyte regression and downregulation of kiss2, gnrh1, fshβ, and lhβ. When the transcript levels of kiss1 and gnrh1 were measured at 4-h intervals in the brain of sapphire devil, a day-high/night-low fluctuation was observed. The hypothalamic-pituitary-gonadal (HPG) axis may be influenced by melatonin, exerting a negative effect at night because the transcript levels of aralkylamine N-acetyltransferase (aanat2) increased during the scotophase. The expression of aanat2 was higher under short-day than long-day conditions, suggesting that there is a seasonal change in melatonin levels at night. It was concluded that change in photoperiod becomes a key factor for controlling the hormone synthesis in the HPG axis through melatonin.

Serotonin modulates melatonin synthesis as an autocrine neurotransmitter in the pineal gland

The pineal gland secretes melatonin principally at night. Regulated by norepinephrine released from sympathetic nerve terminals, adrenergic receptors on pinealocytes activate aralkylamine N-acetyltransferase that converts 5-hydroxytryptamine (5-HT, serotonin) to N-acetylserotonin, the precursor of melatonin. Previous studies from our group and others reveal significant constitutive secretion of 5-HT from pinealocytes. Here, using mass spectrometry, we demonstrated that the 5-HT is secreted primarily via a decynium-22-sensitive equilibrative plasma membrane monoamine transporter instead of by typical exocytotic quantal secretion. Activation of the endogenous 5-HT receptors on pinealocytes evoked an intracellular Ca2+ rise that was blocked by RS-102221, an antagonist of 5-HT2C receptors. Applied 5-HT did not evoke melatonin secretion by itself, but it did potentiate melatonin secretion evoked by submaximal norepinephrine. In addition, RS-102221 reduced the norepinephrine-induced melatonin secretion in strips of pineal gland, even when no exogenous 5-HT was added, suggesting that the 5-HT that is constitutively released from pinealocytes accumulates enough in the tissue to act as an autocrine feedback signal sensitizing melatonin release.

Maternal morphine intake during pregnancy and lactation affects the circadian clock of rat pups

Early-life morphine exposure causes a variety of behavioural and physiological alterations observed later in life. In the present study, we investigated the effects of prenatal and early postnatal morphine on the maturation of the circadian clockwork in the suprachiasmatic nucleus and the liver, and the rhythm in aralkylamine N-acetyltransferase activity in the pineal gland. Our data suggest that the most affected animals were those born to control, untreated mothers and cross-fostered by morphine-exposed dams. These animals showed the highest mesor and amplitude in the rhythm of Per2, Nr1d1 but not Per1 gene expression in the suprachiasmatic nuclei (SCN) and arrhythmicity in AA-NAT activity in the pineal gland. In a similar pattern to the rhythm of Per2 expression in the SCN, they also expressed Per2 in a higher amplitude rhythm in the liver. Five of seven specific genes in the liver showed significant differences between groups in their expression. A comparison of mean relative mRNA levels suggests that this variability was caused mostly by cross-fostering, animals born to morphine-exposed dams that were cross-fostered by control mothers and vice versa differed from both groups of natural mothers raising offspring. Our data reveal that the circadian system responds to early-life morphine administration with significant changes in clock gene expression profiles both in the SCN and in the liver. The observed differences between the groups suggest that the dose, timing and accompanying stress events such as cross-fostering may play a role in the final magnitude of the physiological challenge that opioids bring to the developing circadian clock.

Prolonging photoperiod promotes testosterone synthesis of Leydig cells by directly targeting local melatonin system in rooster testes†.

The study investigated the effects of prolonging photoperiod on the synthesis of testosterone and melatonin in roosters, and the effect of melatonin on testosterone synthesis in rooster Leydig cells as well as its molecular mechanisms. We randomly divided one hundred and twenty 20-week-old roosters into three groups and provided 6, 12.5 and 16h light, respectively. The results showed that prolonging photoperiod promoted testosterone synthesis, decreased melatonin production, and inhibited the expression of melatonin membrane receptors MEL1A, MEL1B, MEL1C, and aralkylamine N-acetyltransferase (AANAT) in rooster testes. Subsequently, rooster Leydig cells were isolated and treated with 0, 0.1, 1, 10, and 100ng/mL melatonin for 36h. The results suggested that melatonin inhibited testosterone synthesis in rooster Leydig cells, and silencing MEL1A and MEL1B relieved the inhibition of melatonin on testosterone synthesis. Additionally, melatonin reduced the intracellular cyclic adenosine monophosphate (cAMP) level and the phosphorylation level of cAMP-response element binding protein (CREB), and CREB overexpression alleviated the inhibition of melatonin on testosterone synthesis. Furthermore, pretreatment with cAMP activator forskolin or protein kinase A (PKA) activator 8-bromo-cAMP blocked the inhibition of melatonin on CREB phosphorylation and testosterone synthesis. These results indicated that prolonging photoperiod promoted testosterone synthesis associated with the decrease in melatonin production and membrane receptors and biosynthetic enzyme of melatonin in rooster testes, and melatonin inhibited testosterone synthesis of rooster Leydig cells by inhibiting the cAMP/PKA/CREB pathway via MEL1A and MEL1B. This may be evidence that prolonging photoperiod could promote testosterone synthesis through the inhibition of the local melatonin pathway in rooster testes.

MiR-375-3p mediates reduced pineal function in hypoxia-ischemia brain damage

The functional roles of microRNAs (miRNAs) have been studied in various diseases, including hypoxic-ischemic brain damage (HIBD). However, changes in the expression of miRNAs and the underlying mechanisms in the pineal gland during HIBD remain unknown. Based on the previous study by microRNA array, hundreds of miRNAs showed altered expression patterns in the pineal gland in a rat model of HIBD. MiR-375-3p was found to be significantly upregulated and abundant in the pineal gland. Further investigation in an in vitro HI model of pinealocytes showed that miRNA-375 exacerbated the damage to pineal function. After oxygen-glucose deprivation / reoxygenation (OGD/R), miR-375-3p expression increased, while aralkylamine N-acetyltransferase (AANAT) expression and melatonin (MT) secretion decreased. Overexpression of miRNA-375 in pinealocytes aggravated the influence of OGD/R on AANAT expression and MT secretion. Because miRNA-375 overexpression in pinealocytes induced decreased rasd1 mRNA and protein expression, rasd1 may mediate the effect of miR-375-3p on pineal function. Furthermore, miR-375-3p aggravated the cognitive impairment caused by HIBD in rats, as observed by Morris water maze test, and also affected emotion and circadian rhythm in HIBD-treated rats. Thus, miR-375-3p may be a key regulatory molecule in the pineal gland following HIBD, and targeting of miR-375-3p may represent a new strategy for the treatment of HIBD.

Draft Genome of the Mirrorwing Flyingfish (Hirundichthys speculiger).

Draft Genome of the Mirrorwing Flyingfish (Hirundichthys speculiger).

Expression characteristics of pineal miRNAs at ovine different reproductive stages and the identification of miRNAs targeting the AANAT gene

BackgroundMany recent studies have shown that miRNAs play important roles in the regulation of animal reproduction, including seasonal reproduction. The pineal gland is a crucial hub in the regulation of seasonal reproduction. However, little is known about the expression characteristics of pineal miRNAs in different reproductive seasons (anestrus and breeding season). Therefore, the expression profiles and regulatory roles of ovine pineal miRNAs were investigated during different reproductive stages using Solexa sequencing technology and dual luciferase reporter assays.ResultsA total of 427 miRNAs were identified in the sheep pineal gland. Significant differences in miRNA expression were demonstrated between anestrus and the breeding season in terms of the frequency distributions of miRNA lengths, number of expressed miRNAs, and specifically and highly expressed miRNAs in each reproductive stage. KEGG analysis of the differentially expressed (DE) miRNAs between anestrus and the breeding season indicated that they are significantly enriched in pathways related to protein synthesis, secretion and uptake. Furthermore, transcriptome analysis revealed that many target genes of DE miRNAs in the ribosome pathway showed relatively low expression in the breeding season. On the other hand, analyses combining miRNA-gene expression data with target relationship validation in vitro implied that miR-89 may participate in the negative regulation of aralkylamine N-acetyltransferase (AANAT) mRNA expression by targeting its 3’UTR at a unique binding site.ConclusionsOur results provide new insights into the expression characteristics of sheep pineal miRNAs at different reproductive stages and into the negative regulatory effects of pineal miRNAs on AANAT mRNA expression.

A de novo Transcriptome Assembly of the European Flounder (Platichthys flesus): The Preselection of Transcripts Encoding Active Forms of Enzymes

The RNA sequencing data sets available for different fish species show a potentially high variety of forms of enzymes just in teleosts. This is primarily considered an effect of the first round of whole-genome duplication with mutations in duplicated genes (isozymes) and alternative splicing of mRNA (isoforms). However, the abundance of the mRNA transcript variants is not necessarily reflected in the abundance of active forms of proteins. We have investigated the transcriptional profiles of two enzymes, aralkylamine N-acetyltransferase (AANAT: EC 2.3.1.87) and N-acetylserotonin O-methyltransferase (ASMT: EC 2.1.1.4), in the eyeball, brain, intestines, spleen, heart, liver, head kidney, gonads, and skin of the European flounder (Platichthys flesus). High-throughput next-generation sequencing technology NovaSeq6000 was used to generate 500M sequencing reads. These were then assembled and filtered producing 75k reliable contigs. Gene ontology (GO) terms were assigned to the majority of annotated contigs/unigenes based on the results of PFAM, PANTHER, UniProt, and InterPro protein database searches. BUSCOs statistics for metazoa, vertebrata, and actinopterygii databases showed that the reported transcriptome represents a high level of completeness. In this article, we show how to preselect transcripts encoding the active enzymes (isozymes or isoforms), using AANAT and ASMT in the European flounder as the examples. The data can be used as a tool to design the experiments as well as a basis for discussion of diversity of enzyme forms and their physiological relevance in teleosts.