Immunocytochemical identification of pinopsin in pineal glands of chicken and pigeon.

Pinopsin is a blue-sensitive photoreceptive molecule possibly involved in photic entrainment of the circadian pacemaker in the chicken pineal gland. To characterize pinopsin as a circadian photoreceptor, antibodies were raised against the C-terminal portion of pinopsin. As expected from the divergence of the amino acid sequence of this region, the resultant antibody cross-reacted with neither chicken rhodopsin nor red-sensitive cone pigment (chicken red). In Western blot analysis, the antibody stained a single band of 42-kDa protein in a detergent-extract of chicken pineal membranes, suggesting that pinopsin (calculated molecular weight, 38187) might be glycosylated and/or palmitoylated. Immunocytochemical examination of pineal sections of the chicken and the pigeon with this antibody revealed strong positive images for most of the membrane structures in the lumen of the follicles. This antibody also stained string- and bulb-shaped structures of the chicken parafollicular cells, the morphology of which resembles those of retinal photoreceptor cells. In contrast to the predominant distribution of pinopsin, a monoclonal antibody specific for chicken red stained a smaller number of membrane structures in the lumen of chicken pineal follicles. These results strongly suggest that the chicken pineal gland contains at least two types of photoreceptive molecules, pinopsin (major) and chicken red (minor). We show that the former molecule is localized in parafollicular pinealocytes and in the outer segments of pinealocytes that make contact with the follicular lumen.

Light-dependent expression of pinopsin gene in chicken pineal gland.

The phase of a circadian clock in the chicken pineal gland is reset by an environmental light signal, which is captured by the pineal photoreceptive molecule(s). Here we show that the mRNA level of pinopsin, a predominant photoreceptive molecule in the pineal gland, undergoes a diurnal fluctuation in chickens maintained on a light/dark cycle. The mRNA levels in the light were approximately six times higher than those in the dark. This fluctuation was not observed in constant darkness, where the mRNA levels remained low. Subsequent light exposure of chickens increased the amount of pinopsin mRNA regardless of the circadian time. Clearly, the expression of pinopsin gene is controlled by a light signal, independent of the circadian clock. In vitro experiments using cultured pineal glands isolated from the visual system also revealed the light-dependent increase in pinopsin mRNA level, indicating that the pineal photoreceptive molecule(s) is responsible for the induction. These results demonstrate the presence of a feedback loop through which the light signal captured by pinopsin stimulates the transcription of its own gene in the chicken pineal gland. In contrast, pinopsin protein remained at an almost constant level in chickens maintained under the same light/dark cycles. The protein level, however, decreased to approximately 50% of the light/dark level under constant darkness and subsequently increased upon exposure to light after the dark period. It is suggested that, under the light/dark cycles, the pinopsin protein level is kept constant by the light-dependent synthesis, which would compensate for a possible degradation of pinopsin in the daytime.

Immunoelectron-microscopic investigation of the subcellular localization of pinopsin in the pineal organ of the chicken.

Pinopsin is a photoreceptive molecule cloned from the chicken pineal organ. An antibody highly specific for pinopsin was applied in light- and electron-microscopic immunocytochemical studies of the pineal organ of 1 to 2-month-old chickens. Intense immunoreactivity was found in the follicular lumen at the light-microscopic level. In addition, small immunoreactive spherical or fibrous structures were diffusely distributed at the parafollicular aspect of the pineal organ. To identify immunoreactive elements precisely, we used pre-embedding immunoelectron microscopy. These studies revealed immunoreactive outer segments of pinealocytes arranged closely side by side in the follicular lumina. The thin initial portion of the outer segment arose from a basal body located in the inner segment. Immunoreactive pear-shaped outer segments occupied small lumina. Follicular lumina displayed immunonegative arrays of whorl-like lamellar membranes. Occasionally, these immunonegative structures were surrounded by immunoreactive concentric lamellar complexes. In the parafollicular pineal parenchyma, long slender cilium-like structures or enlarged cilia and concentric lamellar arrays showed intense immunoreactivity. All immunoreactive structures observed in this study were considered to represent outer segments of pinealocytes of the chicken pineal organ.

Chicken pineal clock genes: implication of BMAL2 as a bidirectional regulator in circadian clock oscillation.

BACKGROUND: In a transcription/translation-based autoregulatory feedback loop of vertebrate circadian clock systems, a BMAL1-CLOCK heterodimer is a positive regulator for the transcription of the negative element gene Per. The chicken pineal gland represents a photosensitive clock tissue, but the pineal clock genes constituting the oscillator loop have been less well characterized. RESULTS: We identified expression of the Per2, Bmal1, Bmal2 and Clock genes in the chicken pineal gland. Messenger RNA levels of these genes exhibited overt circadian rhythms in the pineal cells, both in vivo and in culture. In vitro functional analyses revealed the formation of cBMAL1-cCLOCK and cBMAL2-cCLOCK heteromers. Both of the cBMAL-cCLOCK heteromers activated E-box element-dependent transcription, which was negatively regulated by cPER2 in luciferase assays. Co-expression of cCLOCK, cBMAL1 and cBMAL2 co-operatively activated E-box element-dependent transcription, and a greater level of expression of cBMAL2 inhibited the activation. In the cultured pineal cells, an over-expression of either cBMAL1 or cBMAL2 disrupted the circadian rhythm of melatonin production. CONCLUSION: The functional characterization of the chicken pineal clock molecules supports the key roles of BMAL1, BMAL2 and CLOCK which contribute to the E-box-dependent transcriptional regulation in the circadian clock system.