Pineal gland plays a role in gonadal development after eyelids separation in puppies / La glándula pineal juega un rol en el desarrollo gonadal luego de la separación de los párpados en cachorros

Several functional and morphological studies have been conducted on the pineal gland in many mammalian species; however, no published reports are available on the role of pineal gland on the gonadal development before and after eyelids separation in puppies. Therefore, this study aimed to trace the postnatal histo-morphological changes in the pineal gland and gonads of puppies before (2, 10 and 11 days old) and after (25, 35 and 40 days old) eyelids separation in an attempt to investigate the possible role of pineal gland on the gonadal development. In general, the differentiation of pineal cells, interstitial endocrine cells of testes and stromal ovarian cells coincides with the start of eyelids separation in puppies. Histological examination of stained pineal and gonadal slices of puppies after eyelids separation revealed a remarkable differentiation of pinealocytes and testicular interstitial endocrine cells, as well as presence of some evidence of folliculogenesis in ovary. Surprisingly, melatonin receptor (MT1) protein expression levels were significantly increased in the ovaries and testes of puppies after eyelids separation. Moreover, the mRNA and protein expression of AANAT, a rate-limiting enzyme in melatonin biosynthesis, was notably increased in the pineal gland of opened eyes puppies. Our results suggest an increase of melatonin production from the pineal gland of opened eyes puppies and this could play a vital role in the developmental changes observed in the gonads of these puppies.

Diversos estudios morfológicos y funcionales han sido realizados sobre la glándula pineal en distintas especies de mamíferos. Sin embargo, no hay informes publicados acerca del rol de la glándula pineal en el desarrollo gonadal antes y después de la separación de los párpados en cachorros. Este estudio tuvo como objetivo trazar los cambios histo-morfológicos postnatales en la glándula pineal y las gónadas de los cachorros antes (2, 10 y 11 días de edad) y después (25, 35 y 40 días de edad) de la separación de los párpados, en un intento por investigar el posible rol de la glándula pineal en el desarrollo gonadal. En general, la diferenciación de los pinealocitos, células intersticiales endocrinas de los testículos y las células estromales del ovario coincide con el inicio de la separación de los párpados en cachorros. El examen histológico de glándula pineal y los cortes gonadales de los cachorros, después de la separación de los párpados, reveló una notable diferenciación de los pinealocitos y las células intersticiales endocrinas testiculares, así como la posible evidencia de foliculogénesis en el ovario. Sorprendentemente, en el receptor de melatonina (MT1) los niveles de expresión de proteínas fueron significativamente superiores en los ovarios y los testículos de los cachorros después de la separación de los párpados. Además, el ARNm y la expresión de la proteína AANAT, una enzima limitante de la velocidad en la biosíntesis de la melatonina, aumentaron notablemente en la glándula pineal de los cachorros con los ojos abiertos. Nuestros resultados sugieren que existe un aumento de la producción de melatonina por parte de la glándula pineal en los cachorros con los ojos abiertos, lo que podría jugar un rol vital en los cambios evolutivos observados enlas gónadas de estos cachorros.

Comparison of arylalkylamine N-acetyltransferase and melatonin receptor type 1B immunoreactivity between young adult and aged canine spinal cord.

Melatonin affects diverse physiological functions through its receptor and plays an important role in the central nervous system. In the present study, we compared immunoreactivity patterns of arylalkylamine N-acetyltransferase (AANAT), an enzyme essential for melatonin synthesis, and melatonin receptor type 1B (MT2) in the spinal cord of young adult (2~3 years) and aged (10~12 years) beagle dogs using immunohistochemistry and Western blotting. AANAT-specific immunoreactivity was observed in the nuclei of spinal neurons, and was significantly increased in aged dog spinal neurons compared to young adult spinal neurons. MT2-specific immunoreactivity was found in the cytoplasm of spinal neurons, and was predominantly increased in the margin of the neuron cytoplasm in aged spinal cord compared to that in the young adult dogs. These increased levels of AANAT and MT2 immunoreactivity in aged spinal cord might be a feature of normal aging and associated with a feedback mechanism that compensates for decreased production of melatonin during aging.

Norepinephrine activates NF-κB transcription factor in cultured rat pineal gland.

AIMS: The circadian rhythm in mammalian pineal melatonin secretion is modulated by norepinephrine (NE) released at night. NE interaction with ß1-adrenoceptors activates PKA that phosphorylates the transcription factor CREB, leading to the transcription and translation of the arylalkylamine-N-acetyltransferase (AANAT) enzyme. Several studies have reported the interplay between CREB and the nuclear factor-κB (NF-κB) and a circadian rhythm for this transcription factor was recently described in the rat pineal gland. In this work we studied a direct effect of NE on NF-κB activation and the role played by this factor on melatonin synthesis and Aanat transcription and activity. MAIN METHODS: Cultured rat pineal glands were incubated in the presence of two different NF-κB inhibitors, pyrrolidine-dithiocarbamate or sodium salicylate, and stimulated with NE. Melatonin content was quantified by HPLC with electrochemical detection. AANAT activity was measured by a radiometric assay and the expression of Aanat mRNA was analyzed by real-time PCR. Gel shift assay was performed to study the NF-κB activation in cultured rat pineal glands stimulated by NE. KEY FINDINGS: Our results showed that the p50/p50 homodimer of NF-κB is activated by NE and that it has a role in melatonin synthesis, acting on Aanat transcription and activity. SIGNIFICANCE: Here we present evidence that NF-κB is an important transcription factor that acts, directly or indirectly, on Aanat transcription and activity leading to a modulation of melatonin synthesis. NE plays a role in the translocation of NF-κB p50/p50 homodimer to the nucleus of pinealocytes, thus probably influencing the nocturnal pineal melatonin synthesis.

The arylalkylamine-N-acetyltransferase (AANAT) acetylates dopamine in the digestive tract of goldfish: a role in intestinal motility.

Melatonin has been found in the digestive tract of many vertebrates. However, the enzymatic activity of the arylalkylamine-N-acetyltransferase (AANAT) and the hydroxindole-O-methyltransferase (HIOMT), the last two enzymes of melatonin biosynthesis, have been only measured in rat liver. Therefore, the first objective of the present study is to investigate the functionality of these enzymes in the liver and gut of goldfish, analyzing its possible daily changes and comparing its catalytic properties with those from the retina isoforms. The daily rhythms with nocturnal acrophases in retinal AANAT and HIOMT activities support their role in melatonin biosynthesis. In foregut AANAT activity also show a daily rhythm while in liver and hindgut significant but not rhythmic levels of AANAT activity are found. HIOMT activity is not detected in any of these peripheral tissues suggesting an alternative role for AANAT besides melatonin synthesis. The failure to detect functional HIOMT activity in both, liver and gut, led us to investigate other physiological substrates for the AANAT, as dopamine, searching alternative roles for this enzyme in the goldfish gut. Dopamine competes with tryptamine and inhibits retinal, intestinal and hepatic N-acetyltryptamine production, suggesting that the active isoform in gut is AANAT1. Besides, gut and liver produces N-acetyldopamine in presence of acetyl coenzyme-A and dopamine. This production is not abolished by the presence of folic acid (arylamine N-acetyltransferase inhibitor) in any studied tissue, but a total inhibition occurs in the presence of CoA-S-N-acetyltryptamine (AANAT inhibitor) in liver. Therefore, AANAT1 seems to be an important enzyme in the regulation of dopamine and N-acetyldopamine content in liver. Finally, for the first time in fish we found that dopamine, but not N-acetyldopamine, regulates the gut motility, underlying the broad physiological role of AANAT in the gut.

Association between AA-NAT gene polymorphism and reproductive performance in sheep

Arylalkylamine N-acetyltransferase (AA-NAT) is critical enzyme in Melatonin (MLT) biosynthesis for MLT regulating the animal seasonal breeding. In this study, DNA sequencing methods were applied to detect the polymorphisms of the AA-NAT gene in 179 Chinese sheep belonging to two non-seasonal reproduction breeds and two seasonal reproduction breeds. One mutation at exon 3 (NM_001009461:c.486A > G) was firstly described at the sheep AA-NAT locus. Hence, we described the SmaI PCR-RFLP method for detecting EX3 486A > G mutation, frequencies of the AA-NAT-G allele varied from 0.871 to 0.908 in two non-seasonal reproduction breeds and 0.517 to 0.578 in two seasonal reproduction breeds. The associations of SmaI polymorphism with estrus traits was analyzed in non-seasonal reproduction breeds sheep and seasonal reproduction breeds sheep, the significant statistical results were found between them, the GG genotype frequencies was higher in non-seasonal reproduction breeds (p < 0.001), while, the GA genotype frequencies was higher in seasonal reproduction breeds (p < 0.05). Hence, the EX3 486A > G mutation could facilitate association analysis and serve as a genetic marker for Chinese sheep breeding and genetics.

Current knowledge on the melatonin system in teleost fish.

Melatonin is a much conserved feature in vertebrates that plays a central role in the entrainment of daily and annual physiological rhythms. Investigations aiming at understanding how melatonin mediates the effects of photoperiod on crucial functions and behaviors have been very active in the last decades, particularly in mammals. In fish a clear-cut picture is still missing. Here we review the available data on (i) the sites of melatonin production in fish, (ii) the mechanisms that control its daily and annual rhythms of production and (iii) the characterization of its different receptor subtypes, their location and regulation. The in vivo and in vitro data on melatonin effects on crucial neuroendocrine regulations, including reproduction, growth, feeding and behavioral responses, are also reviewed. Finally we discuss how manipulation of the photic cues impact on fish circannual clock and annual cycle of reproduction, and how this can be used for aquaculture purposes.

Differential resetting process of circadian gene expression in rat pineal glands after the reversal of the light/dark cycle via a 24 h light or dark period transition.

Although studies involving the circadian response to time-zone transitions indicate that the circadian clock usually takes much longer to phase advance than delay, the discrepancy between the circadian resetting induced by photoperiod alteration via a dark or light period transition has yet to be investigated. In mammals, the pineal gland is an important component in the photoneuroendocrine axis, regulating biological rhythms. However, few studies have systematically examined the resetting process of pineal clock-gene expression to date. We investigated the resetting processes of four clock genes (Bmal1, Cry1, Per1, Dec1) and AANAT in the rat pineal gland after the light-dark (LD) reversal via a 24 h light or dark period transition. The resynchronization of the SCN-driven gene AANAT was nearly complete in three days in both situations, displaying similar resetting rates and processes after the differential LD reversals. The resetting processes of the clock genes were characterized by gene-specific, phase-shift modes and differential phase-shift rates between the two different LD reversal modes. The resetting processes of these clock genes were noticeably lengthened after the LD reversal via the light period transition in comparison to via the dark period transition. In addition, among the four examined clock genes, Per1 adjusted most rapidly after the differential LD reversals, while the rhythmic Cry1 expression adjusted most slowly.

Physiology and pharmacology of melatonin in relation to biological rhythms.

Melatonin is an evolutionarily conserved molecule that serves a time-keeping function in various species. In vertebrates, melatonin is produced predominantly by the pineal gland with a marked circadian rhythm that is governed by the central circadian pacemaker (biological clock) in the suprachiasmatic nuclei of the hypothalamus. High levels of melatonin are normally found at night, and low levels are seen during daylight hours. As a consequence, melatonin has been called the "darkness hormone". This review surveys the current state of knowledge regarding the regulation of melatonin synthesis, receptor expression, and function. In particular, it addresses the physiological, pathological, and therapeutic aspects of melatonin in humans, with an emphasis on biological rhythms.

Evolution of the vertebrate pineal gland: the AANAT hypothesis.

The defining feature of the pineal gland is the capacity to function as a melatonin factory that operates on a approximately 24 h schedule, reflecting the unique synthetic capacities of the pinealocyte. Melatonin synthesis is typically elevated at night and serves to provide the organism with a signal of nighttime. Melatonin levels can be viewed as hands of the clock. Issues relating to the evolutionary events leading up to the immergence of this system have not received significant attention. When did melatonin synthesis appear in the evolutionary line leading to vertebrates? When did a distinct pineal gland first appear? What were the forces driving this evolutionary trend? As more knowledge has grown about the pinealocyte and the relationship it has to retinal photoreceptors, it has become possible to generate a plausible hypothesis to explain how the pineal gland and the melatonin rhythm evolved. At the heart of the hypothesis is the melatonin rhythm enzyme arylalkylamine N-acetyltransferase (AANAT). The advances supporting the hypothesis will be reviewed here and expanded beyond the original foundation; the hypothesis and its implications will be addressed.

Starting the zebrafish pineal circadian clock with a single photic transition.

The issue of what starts the circadian clock ticking was addressed by studying the developmental appearance of the daily rhythm in the expression of two genes in the zebrafish pineal gland that are part of the circadian clock system. One encodes the photopigment exorhodopsin and the other the melatonin synthesizing enzyme arylalkylamine N-acetyltransferase (AANAT2). Significant daily rhythms in AANAT2 mRNA abundance were detectable for several days after fertilization in animals maintained in a normal or reversed lighting cycle providing 12 h of light and 12 h of dark. In contrast, these rhythms do not develop if animals are maintained in constant lighting or constant darkness from fertilization. In contrast to exorhodopsin, rhythmicity of AANAT2 can be initiated by a pulse of light against a background of constant darkness, by a pulse of darkness against a background of constant lighting, or by single light-to-dark or dark-to-light transitions. Accordingly, these studies indicate that circadian clock function in the zebrafish pineal gland can be initiated by minimal photic cues, and that single photic transitions can be used as an experimental tool to dissect the mechanism that starts the circadian clock in the pineal gland.

A possible new role for fish retinal serotonin-N-acetyltransferase-1 (AANAT1): Dopamine metabolism.

Serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase, AANAT) is the key enzyme in the generation of melatonin rhythms in the pineal gland and retinal photoreceptors. Rhythmic AANAT activity drives rhythmic melatonin production in these tissues. Two AANATs, AANAT1 and AANAT2, are present in teleost fish species. Different spatial expression patterns, enzyme kinetics and substrate preferences suggest that they may have different functions. Enzyme activity assays revealed that recombinant seabream and zebrafish AANAT1s, but not AANAT2s, acetylate dopamine with kinetic characteristics that are similar to those for tryptamine acetylation. High performance liquid chromatography analysis of seabream retinal extracts indicated the presence of N-acetyldopamine. Time-of-day analysis of retinal AANAT activity and concentration of melatonin, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and N-acetyldopamine revealed a daily pattern of retinal melatonin and N-acetyldopamine production that are correlated with retinal AANAT1 activity. In situ hybridization analysis of seabream retinal sections indicated that tyrosine hydroxylase is expressed in the inner nuclear layer (INL) and that AANAT1 is expressed in the outer nuclear layer (ONL) and INL. Together, these observations point to the possibility that dopamine is acetylated by retinal AANAT1 in the INL. Such novel activity of AANAT1 may reflect an important function in the circadian physiology of the retina.

The candidate gene, Clock, localizes to a strong spawning time quantitative trait locus region in rainbow trout.

We applied a candidate gene mapping approach to an existing quantitative trait loci (QTL) data set for spawning date in rainbow trout (Oncorynchus mykiss) to ascertain whether these genes could potentially account for any observed QTL effects. Several genes were chosen for their known or suspected roles in reproduction, circadian, or circannual timing, including salmon-type gonadotropin-releasing hormone 3A and 3B (GnRH3A and GnRH3B), Clock, Period1, and arylalkylamine N-acetlytransferase-1 and -2 (AANAT-1 and AANAT-2). Genes were sequenced, and polymorphisms were identified in parents of two rainbow trout mapping families, one of which was used previously to detect spawn timing QTL. Interval mapping was used to identify associations between genetic markers and spawning date effects. Using a genetic map that was updated with 574 genetic markers (775 total), we found evidence for 11 significant or suggestive QTL regions. Most QTL were only localized within one of the parents; however, a strong QTL region was identified in both female and male parents on linkage group RT-8 that explained 20% and 50% of trait variance, respectively. The Clock gene mapped to this region. Period1 mapped to a region in the female parent associated with a marginal effect (P = .056) on spawn timing. Other candidate genes were not associated with significant QTL effects.

Cell culture models for oscillator and pacemaker function: recipes for dishes with circadian clocks?

Primary cell cultures of avian pinealocytes and the mammalian suprachiasmatic nucleus (SCN), immortalized cell lines derived from the SCN (SCN2.2), and fibroblasts derived from mice and rats have been employed as in vitro models to study the cellular and molecular mechanisms underlying circadian biological clocks. This article compares and contrasts these model systems and describes methods for avian pinealocyte cultures, immortalized SCN2.2 cells, and mouse fibroblast culture. Each of these culture models has advantages and disadvantages. Avian pinealocytes are photoreceptive, contain a circadian pacemaker, and produce rhythms of an easily assayed endocrine output-melatonin. However, the molecular mechanisms underlying pinealocyte function are not understood. SCN2.2 cells express metabolic and molecular rhythms and can impose rhythmicity on cocultured cells as well as rat behavior when transplanted into the brain. Yet, the entrainment pathways are not experimentally established in these cells. Fibroblast cultures are simple to produce and express molecular clock gene rhythms, but they express neither physiological rhythmicity nor pacemaker properties. The relative merits of these culture systems, as well as their impact on understanding circadian organization in vivo, are also considered.