Jérôme Guerlotté

An antiserum against chicken hydroxyindole-O-methyltransferase reacts with the enzyme from pineal gland and retina and labels pineal modified photoreceptors

Biosynthesis of the indolic hormone melatonin has been reported in the pineal gland and retina. The terminal step of melatonin synthesis is catalysed by hydroxyindole-O-methyltransferase (HIOMT), an enzyme displaying highest levels of activity in the pineal gland and retina. Several laboratories have suggested that melatonin synthesis might take place in retinal photoreceptors and in photoreceptor-derived cells of the pineal gland. Experimental support to this hypothesis is progressively building up with the immunocytochemical identification of HIOMT-containing cells in various animal species. In the present report, HIOMT was purified from the chicken pineal gland using a one-step chromatographic procedure and an antiserum against the enzyme was obtained in the rabbit. The antiserum was further purified by immunoadsorption chromatography on chicken brain proteins. Using electroblots immunochemical labeling, HIOMT from chicken pineal gland and retina was identified as a 38-kDa protein. Pineal HIOMT was further resolved into components of different pHi-values (5.4-5.7 and 6.8), using two-dimensional gel electrophoresis. Immunoprecipitation of HIOMT activity was observed in pineal homogenates and, for the first time, in homogenates of the retina. Immunofluorescence microscopy provided the first evidence that HIOMT is contained in modified photoreceptors of the chicken pineal gland. No immunofluorescence could be observed in the retina, maybe due to the lower level of HIOMT activity in this tissue. Together, the data indicate that the antiserum may be a useful tool to study the regulation of HIOMT synthesis in the pineal gland and in the retina. Further work is required to identify HIOMT-containing cells in the retina.

Pineal-retinal molecular relationships: distribution of S-antigen in the pineal complex

The immunocytochemical localization of S-antigen, a specific protein first discovered in retinal photoreceptors, was studied in the pineal complex of vertebrates (eel, pike, frog, lizard, passerines, mouse, hamster) using monoclonal antibody immunofluorescence. S-antigen immunoreactivity was demonstrated concurrently in retinal photoreceptors and in most pineal phototransducers of all species, i.e. in pineal cells of the receptor series (cone-like, modified photoreceptor cells, pinealocytes) and in cone-like photoreceptors of the frog frontal organ and lizard parietal eye. The labelling was distributed either in all compartments of these cells, or restricted to outer segments. The functional significance of the S-antigen as well as some phylogenetic and ontogenic implication of this marker are discussed.

Hydroxyindole-O-methyltransferase in the Chicken Retina: Immunocytochemical- Localization and Daily Rhythm of mRNA

In the vertebrate retina and pineal gland, melatonin production displays diurnal variations with high levels at night. Hydroxyindole‐O‐methyltransferase (HIOMT, EC 2.1.1.4) catalyses the last step of melatonin biosynthesis. In the present study, a cDNA encoding chicken HIOMT was used to examine the effects of environmental lighting on HIOMT mRNA expression in the chicken retina. A day/night rhythm of HIOMT mRNA level was observed, with an average 5‐fold increase during the night. Light strongly suppressed the night‐time rise in HIOMT mRNA concentration while darkness prevented its daytime fall. An antibody directed against chicken HIOMT was used for immunocytochemical identification of retinal melatoninergic cells. HIOMT immunoreactivity could be observed in rods as well as in cones. However, the lowest levels of HIOMT immunoreactivity were always observed in the accessory cones of double cones. A few HIOMT‐positive cell bodies could also be observed in the inner nuclear layer. Altogether, these data indicate that HIOMT gene expression in the retina is organized on a daily basis as a direct response to light, and that the different types of photoreceptors may not be equally involved in melatonin production.

Molecular and cellular aspects of hydroxyindole-O-methyltransferase expression in the developing chick pineal gland

The pineal gland influences circadian activity and seasonal breeding through the production of an indolic hormone, melatonin. The terminal step of melatonin biosynthesis is catalyzed by hydroxyindole-O-methyltransferase (HIOMT). Using an antibody directed against HIOMT, we examined the differentiation of the melatoninergic phenotype in the developing chick pineal gland. HIOMT first appeared 4 days before hatch and rose linearly until the 7th day posthatch. This was correlated with an increased immunoreactivity of the 38 kDa enzyme on Western blots and with an accelerated rate of HIOMT biosynthesis as demonstrated by [35S]methionine labeling. Immunocytochemistry revealed a growing number of HIOMT-positive cells between day 2 before hatch and day 15 posthatch. Until hatching HIOMT was expressed almost exclusively in modified photoreceptors. Parafollicular pinealocytes became HIOMT-positive mostly after hatching. Their different timings of functional differentiation emphasize the existence of two populations of melatonin-producing cells in the chick pineal gland.

Synthesis of melatonin by the pineal modified photoreceptors of birds immunocytochemical localization of hydroxyindole-O-methyltransferase.

Photoperiodic control of several biological rhythms is exerted through the inhibitory effect of light on melatonin synthesis in the pineal organ. Hydroxyindole‐O‐methyltransferase (HIO‐MT), the last acting‐enzyme in melatonin biosynthesis, constitutes a specific marker of melatoninergic cells. In the present study, an antibody directed against chicken HIOMT was affinity‐purified and used to identify melatoninergic cells in the pineal organ of chicken, quail, sparrow and blackbird. Regardless of the species, intense immunocytochemical reactions were observed in modified photoreceptors, whereas other cellular constituents (mostly glial cells) remained unlabeled. We conclude that modified photoreceptors synthesize melatonin in the avian pineal gland and are thus accountable for the translation of the photoperiodic input into hormonal output.

Hydroxyindole-O-methyltransferase gene expression in the pineal gland of chicken embryo: development of messenger RNA levels and regulation by serum

Hydroxyindole-O-methyltransferase (HIOMT), the enzyme which catalyzes the final step of melatonin biosynthesis, constitutes a marker of the functional differentiation of pineal cells. In addition, a day/night rhythm of HIOMT mRNA concentration, previously described in the chicken pineal gland [6], would suggest that HIOMT gene transcription is one output of the circadian system that controls pineal function. The study sought to monitor the developmental expression of HIOMT mRNA in the chick pineal gland and to investigate a possible role of instructive signals in this differentiation process. RT-PCR analysis indicated that HIOMT mRNA is expressed at embryonic day 8 (E8). At E12, HIOMT mRNA became detectable on northern blots and traces of HIOMT activity could be measured. HIOMT mRNA concentration increased 100-fold between E14 and day 10 post-hatch, then levelled off. A day/night rhythm of HIOMT mRNA concentration was readily observed in the pineal gland of 2-day-old chicks. Pineal glands isolated on minimum culture medium at E11 stopped developing HIOMT gene expression. However, the addition of serum to the culture medium restored HIOMT mRNA concentration to the levels observed in vivo. The data suggest that the functional differentiation of melatoninergic cells observed during the second week of embryonic life may be controlled [correction of controled] by serum factors.

Cellular localization of hydroxyindole-O-methyltransferase mRNA in the chicken pineal gland.

Hydroxyindole-O-methyltransferase (HIOMT), is the last enzyme of the melatonin biosynthesis pathway. We have applied immunocytochemistry and in situ hybridization to localize HIOMT producing cells, in the chicken epithalamus including the pineal gland. The selectivity of the hybridization signal over melatoninergic cells was ascertained by comparison with the immunolabelling. HIOMT transcripts could be observed in the two cell layers of the pineal follicles and in the pineal stalk, but not in other epithalamic regions. These results indicate that pineal modified photoreceptors and parafollicular pinealocyte-like cells represent the two populations of melatonin-producing cells of the chicken epithalamus.

Long-term effects of constant light or darkness on chicken pineal hydroxyindole-O-methyltransferase expression: biochemical and cellular aspects

Summary1.Chickens kept in constant light, as opposed to constant darkness, display a twofold increase in the activity of pineal hydroxyindole-O-methyltransferase (HIOMT), the last acting enzyme in the melatonin pathway.2.Using an immunological approach, we presently show that this regulation of HIOMT activity reflects changes in the concentration of a single molecular form of the enzyme protein (a 38 kDa polypeptide). Immunohistofluorescence indicates that these concentration changes concurrently affect modified photoreceptors and pinealocyte-like cells in the chicken pineal organ.3.Together, the present data support the hypothesis that environmental lighting might regulate the expression of the HIOMT gene.

Cyclic AMP increases hydroxyindole-O-methyltransferase mRNA levels in the chicken pineal gland, but is not required for circadian rhythmicity of this transcript

Hydroxyindole-O-methyltransferase (HIOMT) plays an important role as the final enzyme in the synthesis of melatonin. In the chicken pineal gland, HIOMT mRNA concentration exhibits a circadian rhythm with a threefold peak at midday. The present study sought to evaluate the possible role of cyclic AMP in this transcriptional rhythm. In cultured pineal glands from 4-day-old chicks, cyclic AMP analogs and the adenylate cyclase activator, forskolin, increased HIOMT mRNA levels twofold to threefold in a dose-dependent manner. Vasoactive intestinal polypeptide increased HIOMT mRNA levels by 50%. Actinomycin-D chase experiments indicated that cyclic AMP did not affect the stability of HIOMT mRNA, thus providing indirect evidence that the effect of cyclic AMP was exerted at transcriptional level. In cultured pineal glands from 11 days embryos, HIOMT mRNA levels failed to respond to cyclic AMP. However, a daily rhythm of HIOMT mRNA, with an endogenous component in constant darkness was clearly observed at this developmental stage. This observation indicates that cyclic AMP is not required for circadian rhythmicity of HIOMT gene transcription.