Laurence Besseau

Melatonin effects on the hypothalamo–pituitary axis in fish

Melatonin, a hormonal output signal of vertebrate circadian clocks, contributes to synchronizing behaviors and neuroendocrine regulations with the daily and annual variations of the photoperiod. Conservation and diversity characterize the melatonin system: conservation because its pattern of production and synchronizing properties are a constant among vertebrates; and diversity because regulation of both its synthesis and modes of action have been profoundly modified during vertebrate evolution. Studies of the targets and modes of action of melatonin in fish, and their parallels in mammals, are of interest to our understanding of time-related neuroendocrine regulation and its evolution from fish to mammals, as well as for aquacultural purposes.

Structural aspects of fish skin collagen which forms ordered arrays via liquid crystalline states

The ability of acid-soluble type I collagen extracts from Soleidae flat fish to form ordered arrays in condensed phases has been compared with data for calf skin collagen. Liquid crystalline assemblies in vitro are optimized by preliminary treatment of the molecular population with ultrasounds. This treatment requires the stability of the fish collagen triple helicity to be controlled by X-ray diffraction and differential scanning calorimetry and the effect of sonication to be evaluated by viscosity measurements and gel electrophoresis. The collagen solution in concentrations of at least 40 mg ml(-1) showed in polarized light microscopy birefringent patterns typical of precholesteric phases indicating long-range order within the fluid collagen phase. Ultrastructural data, obtained after stabilization of the liquid crystalline collagen into a gelated matrix, showed that neutralized acid-soluble fish collagen forms cross-striated fibrils, typical of type I collagen, following sine wave-like undulations in precholesteric domains. These ordered geometries, approximating in vivo situations, give interesting mechanical properties to the material.

Liquid crystalline assemblies of collagen in bone and in vitro systems

Precise descriptions of the three-dimensional arrangements of collagen in bone are essential to understand the mechanical properties of this complex tissue. Transmission electron microscopy (TEM) analysis of decalcified human compact bone in section reveals characteristic patterns forming regular series of nested arcs. Such patterns are a direct consequence of an organization described as a twisted plywood and relate the distribution of collagen fibrils in osteons with that of molecules in cholesteric liquid crystals. The hypothesis that liquid crystalline properties are involved in the morphogenesis of dense collagen matrices was supported by data obtained in vitro. At a molecular level, acid-soluble collagen molecules spontaneously assemble, at concentrations of 50mg/ml or more, in precholesteric-banded patterns and cholesteric phases, identified by polarized light microscopy. In a more physiological context, these results were conforted, with the precursor molecule of collagen, procollagen, soluble at neutral pH. This protein spontaneously forms liquid crystalline precholesteric phases corresponding to banded patterns and birefringent cords. Stabilization of the liquid crystalline collagen, induced by pH modification and fibril formation, shows characteristic morphologies in TEM, which directly mimic arrays described in vivo. Undulating fibrils are indeed similar to crimp morphologies described in tendons and continuously twisting fibrils, and give rise to arced patterns similar to supra-molecular architectures identified in compact bone.

Structural and Functional Evolution of the Pineal Melatonin System in Vertebrates

In most species daily rhythms are synchronized by the photoperiodic cycle. They are generated by the circadian system, which is made of a pacemaker, an entrainment pathway to this clock, and one or more output signals. In vertebrates, melatonin produced by the pineal organ is one of these outputs. The production of this time‐keeping hormone is high at night and low during the day. Despite the fact that this is a well‐preserved pattern, the pathways through which the photoperiodic information controls the rhythm have been profoundly modified from early vertebrates to mammals. The photoperiodic control is direct in fish and frogs and indirect in mammals. In the former, full circadian systems are found in photoreceptor cells of the pineal organ, retina, and possibly brain, thus forming a network where melatonin could be a hormonal synchronizer. In the latter, the three elements of a circadian system are scattered: the photoreceptive units are in the eyes, the clocks are in the suprachiasmatic nuclei of the hypothalamus, and the melatonin‐producing units are in the pineal cells. Intermediate situations are observed in sauropsids. Differences are also seen at the level of the arylalkylamine N‐acetyltransferase (AANAT), the enzyme responsible for the daily variations in melatonin production. In contrast to tetrapods, teleost fish AANATs are duplicated and display tissue‐specific expression; also, pineal AANAT is special—it responds to temperature in a species‐specific manner, which reflects the fish ecophysiological preferences. This review summarizes anatomical, structural, and molecular aspects of the evolution of the melatonin‐producing system in vertebrates.

Production of ordered collagen matrices for three-dimensional cell culture

The aim of this study was to produce collagen gels with controlled fibrillar order as matrices for cell culture. Their structural characterization and colonization by human dermal fibroblasts arc presently reported. Ordered matrices are obtained by using the property of type I collagen monomers to self-assemble in liquid crystalline arrays by slow evaporation of acidic solutions at high concentrations. Induction of fibrillogenesis concomittent with the stabilization of the supramolecular order is then obtained, within petri dishes, by gelation of the viscous preparations under ammoniac vapours. For comparison, dermal equivalents, in which collagen compaction depends on fibroblasts contraction, are made according to the method of Bell et al. (Proc. Natl. Acad. Sci. 76(3) (1979) 1274). The fibrillar arrangement of the collagen network in the samples is determined by polarizing optical microscopy and by transmission electron microscopy. Whereas dermal equivalents exhibit heterogeneous distributions of fibrils, two differents types of order are obtained in the stabilized liquid crystalline collagen samples, namely aligned, i.e. nematic, at 20 mg/ml, or crimped, i.e. precholesteric, at 40 mg/ml. The morphology and behaviour of fibroblasts seeded on the surface of the matrices are analysed from day 1 to day 21. The cells are viable, proliferate at the surface of ordered matrices and migrate up to 400 microm in depth. Production of concentrated and ordered collagen matrices provides new perspectives to study the behaviour of cells in a valorized three-dimensional context where the fibrillar organization becomes close to in vivo situations.

Cloning and retinal expression of melatonin receptors in the European sea bass, Dicentrarchus labrax

Melatonin contributes to synchronizing behaviors and physiological functions to daily and seasonal rhythm in fish. However, no coherent vision emerges because the effects vary with the species, sex, age, moment of the year or sexual cycle. And, scarce information is available concerning the melatonin receptors, which is crucial to our understanding of the role melatonin plays. We report here the full length cloning of three different melatonin receptor subtypes in the sea bass Dicentrarchus labrax, belonging, respectively, to the MT1, MT2 and Mel1c subtypes. MT1, the most abundantly expressed, was detected in the central nervous system, retina, and gills. MT2 was detected in the pituitary gland, blood cells and, to a lesser extend, in the optic tectum, diencephalon, liver and retina. Mel1c was mainly expressed in the skin; traces were found in the retina. The cellular sites of MT1 and MT2 expressions were investigated by in situ hybridization in the retina of pigmented and albino fish. The strongest signals were obtained with the MT1 riboprobes. Expression was seen in cells also known to express the enzymes of the melatonin biosynthesis, i.e., in the photoreceptor, inner nuclear and ganglion cell layers. MT1 receptor mRNAs were also abundant in the retinal pigment epithelium. The results are consistent with the idea that melatonin is an autocrine (neural retina) and paracrine (retinal pigment epithelium) regulator of retinal function. The molecular tools provided here will be of valuable interest to further investigate the targets and role of melatonin in nervous and peripheral tissues of fish.

Drastic neofunctionalization associated with evolution of the timezyme AANAT 500 Mya

Significance The pineal gland is dedicated to the production of melatonin. Submammalian pineal glands can also detect light, and the retinas of many species can make melatonin. From this finding and others, it is seems that both tissues evolved from a common ancestral photodetector. A key factor driving their independent evolution may have been the evolution of melatonin synthesis and more specifically, the timezyme, a form of arylalkylamine N-acetyltransferase (AANAT) that plays a key role in controlling rhythmic production of melatonin. The current report indicates that the timezyme evolved from a primitive form of AANAT over 500 Mya in chordate evolution through a process of gene duplication followed by rapid neofunctionalization and that it was not a posthoc acquisition. Melatonin (N-acetyl-5-methoxytrypamine) is the vertebrate hormone of the night: circulating levels at night are markedly higher than day levels. This increase is driven by precisely regulated increases in acetylation of serotonin in the pineal gland by arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme in the synthesis of melatonin. This unique essential role of AANAT in vertebrate timekeeping is recognized by the moniker the timezyme. AANAT is also found in the retina, where melatonin is thought to play a paracrine role. Here, we focused on the evolution of AANAT in early vertebrates. AANATs from Agnathans (lamprey) and Chondrichthyes (catshark and elephant shark) were cloned, and it was found that pineal glands and retinas from these groups express a form of AANAT that is compositionally, biochemically, and kinetically similar to AANATs found in bony vertebrates (VT-AANAT). Examination of the available genomes indicates that VT-AANAT is absent from other forms of life, including the Cephalochordate amphioxus. Phylogenetic analysis and evolutionary rate estimation indicate that VT-AANAT evolved from the nonvertebrate form of AANAT after the Cephalochordate–Vertebrate split over one-half billion years ago. The emergence of VT-AANAT apparently involved a dramatic acceleration of evolution that accompanied neofunctionalization after a duplication of the nonvertebrate AANAT gene. This scenario is consistent with the hypotheses that the advent of VT-AANAT contributed to the evolution of the pineal gland and lateral eyes from a common ancestral photodetector and that it was not a posthoc recruitment.

Melatonin receptors in the brain of the European sea bass: An in situ hybridization and autoradiographic study.

Melatonin is synthesized in the pineal organ and retina of vertebrates and exhibits a clear nocturnal rhythm of secretion. This hormone influences a number of important physiological processes acting through specific transmembrane G‐protein‐coupled receptors. Recently, we have cloned three different melatonin receptors in sea bass belonging to the MT1, MT2, and Mel1c subtypes. In this paper, we have analyzed the central expression of the MT1 gene by in situ hybridization and compared its distribution with the localization of 2‐[125I]‐iodomelatonin binding sites. In situ hybridization and autoradiographic studies provided consistent results. Melatonin receptors were mainly expressed in visually related areas of the sea bass brain, such as the pretectal area, glomerular complex, optic tectum, torus longitudinalis, and thalamus. A conspicuous expression was also detected in neuroendocrine regions including the ventral telencephalon, preoptic area, and hypothalamus. Furthermore, melatonin receptors were evident in the ganglionic cell layer of the cerebellum. The presence of iodomelatonin binding and/or MT1 mRNA‐expressing cells was also observed in the hindbrain, in particular in the oculomotor and trigeminal nuclei and in the reticular formation. Our results suggest an important role of MT1 in the mediation of melatonin actions in visual/light integration, mechanoreception, somatosensation, eye‐body motor coordination, and integrative and neuroendocrine functions. Remarkable differences in the number and distribution of brain nuclei expressing MT1 mRNAs in sea bass and trout, the only fish species analyzed to date, represent another piece of evidence for differences in the organization of the visual and circadian systems observed between salmoniform and perciform teleosts. J. Comp. Neurol. 518:3495–3511, 2010.

Banded patterns in liquid crystalline phases of type I collagen: relationship with crimp morphology in connective tissue architecture.

Solutions of type I acid soluble collagen were studied in light and electron microscopy at concentrations over 40 mg/ml. Banded patterns spontaneously emerge in samples observed between crossed polars between slide and coverslip. The textures are interpreted as precholesteric, appearing at the transition between the isotropic phases, due to random molecular order, and the cholesteric phase corresponding to a highly organized three-dimensional structure. Type I collagen banded patterns correspond to regular undulations of the molecular directions with an observed periodicity in the range of 1 to 10 microm. This interpretation is verified by ultrastructural analysis of precholesteric samples gelled under ammonium vapors. Results are discussed in regard to banded patterns described either within synthetic polymer systems or within collagen extracellular matrices. Self-assembled liquid crystalline phases of collagen generate crimp morphologies. Their possible relationship with early secretion steps in the development of connective tissues is discussed.

Molecular and Cellular Regulation of Pineal Organ Responses

Publisher Summary This chapter focuses on the structural and functional properties of the fish pineal organ, and on the molecular mechanisms, that contribute to the synthesis of rhythmic output signals, including melatonin, the timekeeping molecule of the organism. The pineal gland of fish is an evagination of the roof of the diencephalon, which locates in a window below the skull. The structural characteristics and functional properties of the pineal photoreceptors classify them into the cone family of photoreceptor cells, as found in the retina. The pineal epithelium is organized like a retina that would contain cone‐like photoreceptor cells, connecting to second‐order neurons. Supporting “interstitial cells” would be the homologous of the retinal Muller cells. The fish pineal gland is a nonvisual photosensitive organ that transduces the light information and elaborates nervous and neurohormonal messages in response to changes in illumination—the nervous message is an excitatory neurotransmitter and the hormonal message is melatonin. The main function of the fish pineal organ is to integrate light information and elaborate messages that will affect the animals physiology. The photoreceptor cells occupy a key position because they are at the interface between the environment and the organism providing rapid (time scale of seconds) and less rapid (time scale of several hours) responses to environmental light.