Pituitary Hormones mRNA Abundance in the Mediterranean Sea Bass Dicentrarchus labrax: Seasonal Rhythms, Effects of Melatonin and Water Salinity.

In fish, most hormonal productions of the pituitary gland display daily and/or seasonal rhythmic patterns under control by upstream regulators, including internal biological clocks. The pineal hormone melatonin, one main output of the clocks, acts at different levels of the neuroendocrine axis. Melatonin rhythmic production is synchronized mainly by photoperiod and temperature. Here we aimed at better understanding the role melatonin plays in regulating the pituitary hormonal productions in a species of scientific and economical interest, the euryhaline European sea bass Dicentrarchus labrax. We investigated the seasonal variations in mRNA abundance of pituitary hormones in two groups of fish raised one in sea water (SW fish), and one in brackish water (BW fish). The mRNA abundance of three melatonin receptors was also studied in the SW fish. Finally, we investigated the in vitro effects of melatonin or analogs on the mRNA abundance of pituitary hormones at two times of the year and after adaptation to different salinities. We found that (1) the reproductive hormones displayed similar mRNA seasonal profiles regardless of the fish origin, while (2) the other hormones exhibited different patterns in the SW vs. the BW fish. (3) The melatonin receptors mRNA abundance displayed seasonal variations in the SW fish. (4) Melatonin affected mRNA abundance of most of the pituitary hormones in vitro; (5) the responses to melatonin depended on its concentration, the month investigated and the salinity at which the fish were previously adapted. Our results suggest that the productions of the pituitary are a response to multiple factors from internal and external origin including melatonin. The variety of the responses described might reflect a high plasticity of the pituitary in a fish that faces multiple external conditions along its life characterized by marked daily and seasonal changes in photoperiod, temperature and salinity.

Circadian rhythms in Mexican blind cavefish Astyanax mexicanus in the lab and in the field.

Biological clocks have evolved as an adaptation to life on a rhythmic planet, synchronising physiological processes to the environmental light-dark cycle. Here we examine circadian clock function in Mexican blind cavefish Astyanax mexicanus and its surface counterpart. In the lab, adult surface fish show robust circadian rhythms in per1, which are retained in cave populations, but with substantial alterations. These changes may be due to increased levels of light-inducible genes in cavefish, including clock repressor per2. From a molecular standpoint, cavefish appear as if they experience 'constant light' rather than perpetual darkness. Micos River samples show similar per1 oscillations to those in the lab. However, data from Chica Cave shows complete repression of clock function, while expression of several light-responsive genes is raised, including DNA repair genes. We propose that altered expression of light-inducible genes provides a selective advantage to cavefish at the expense of a damped circadian oscillator.

Circadian clock regulation of the cell cycle in the zebrafish intestine.

The circadian clock controls cell proliferation in a number of healthy tissues where cell renewal and regeneration are critical for normal physiological function. The intestine is an organ that typically undergoes regular cycles of cell division, differentiation and apoptosis as part of its role in digestion and nutrient absorption. The aim of this study was to explore circadian clock regulation of cell proliferation and cell cycle gene expression in the zebrafish intestine. Here we show that the zebrafish gut contains a directly light-entrainable circadian pacemaker, which regulates the daily timing of mitosis. Furthermore, this intestinal clock controls the expression of key cell cycle regulators, such as cdc2, wee1, p21, PCNA and cdk2, but only weakly influences cyclin B1, cyclin B2 and cyclin E1 expression. Interestingly, food deprivation has little impact on circadian clock function in the gut, but dramatically reduces cell proliferation, as well as cell cycle gene expression in this tissue. Timed feeding under constant dark conditions is able to drive rhythmic expression not only of circadian clock genes, but also of several cell cycle genes, suggesting that food can entrain the clock, as well as the cell cycle in the intestine. Rather surprisingly, we found that timed feeding is critical for high amplitude rhythms in cell cycle gene expression, even when zebrafish are maintained on a light-dark cycle. Together these results suggest that the intestinal clock integrates multiple rhythmic cues, including light and food, to function optimally.

Cyclin-dependent kinase inhibitor p20 controls circadian cell-cycle timing.

Specific stages of the cell cycle are often restricted to particular times of day because of regulation by the circadian clock. In zebrafish, both mitosis (M phase) and DNA synthesis (S phase) are clock-controlled in cell lines and during embryo development. Despite the ubiquitousness of this phenomenon, relatively little is known about the underlying mechanism linking the clock to the cell cycle. In this study, we describe an evolutionarily conserved cell-cycle regulator, cyclin-dependent kinase inhibitor 1d (20 kDa protein, p20), which along with p21, is a strongly rhythmic gene and directly clock-controlled. Both p20 and p21 regulate the G1/S transition of the cell cycle. However, their expression patterns differ, with p20 predominant in developing brain and peak expression occurring 6 h earlier than p21. p20 expression is also p53-independent in contrast to p21 regulation. Such differences provide a unique mechanism whereby S phase is set to different times of day in a tissue-specific manner, depending on the balance of these two inhibitors.

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.