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The scornea and sclera have been shown to exhibit circadian rhythms in cellular proliferation, wound healing and extracellular matrix synthesis. The distribution of melatonin Mel1a and Mel1c receptors was examined in the cornea and sclera of the Xenopus laevis eye in order to determine whether melatonin may potentially influence the growth and/or development of these ocular tissues. Sections of adult X. laevis eyes were analyzed by immunocytochemistry and confocal microscopy, using antibodies prepared against specific peptide sequences of the Xenopus Mel1a and Mel1c receptor proteins. Antibodies were pre- incubated with their appropriate antigenic peptides to control for non-specific labelling. Analysis of the distribution of Mel1a and Mel1c receptor immunoreactivity in the Xenopus eye revealed that both the Mel1a and Mel1c receptors were located in the outer fibrous layer (OFL) of the sclera, with Mel1c labelling being the most prominent. Similarly, Mel1a and Mel1c (Mel1c mostly) were also located in cells of the inner fibrous layer (IFL) with Mel1c being most abundant. The chondrocytes of the cartilaginous layer also appeared to express Mel1a, Mel1c, or both receptors. Both Mel1a and Mel1c receptor immunoreactivity were observed in the corneal epithelium and endothelium. Whereas the Mel1a antibody labelled the entire corneal epithelial layer, the Mel1c antibody labelled only the most superficial layer of epithelial cells. Cell processes of fibroblasts of the corneal stroma were immunoreactive for either Mel1a or Mel1c receptors. The identification of Mel1a and Mel1c receptors in restricted distributions in the cornea and sclera suggests that melatonin may play a role in the cellular physiology of these ocular tissues.
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12873453
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Fig. 1.
Immunocytochemistry of Xenopus laevis cornea with melatonin Mel1a and Mel1c receptor antibodies. (A) Corneal section stained with blue nuclear dye (DAPI). (B) Corneal section incubated with Mel1a receptor antibody followed by incubation in secondary antibody conjugated to a red fluorescent dye. Mel1a labelling is intense in corneal epithelium (arrowheads) and endothelium, and to a lesser extent labels cells in the corneal stroma. (C) Corneal section incubated with Mel1c receptor antibody followed by incubation in secondary antibody conjugated to a green fluorescent dye. Mel1c labelling is intense in the superficial layer of corneal epithelium and in cells of the corneal stroma. The corneal endothelium and deeper layers of epithelium (arrowheads) were weakly stained. (D) Merged image of Mel1a and Mel1c labelled cornea. Yellow color indicates areas of co- localization of Mel1a and Mel1c. Scale bar=100 μm.
Fig. 2.
Immunocytochemistry of Xenopus laevis sclera with melatonin Mel1a and Mel1c receptor antibodies. (A) Scleral section stained with blue nuclear dye (DAPI). (B) Scleral section incubated with Mel1a receptor antibody followed by incubation in secondary antibody conjugated to a red fluorescent dye. Mel1a labelling is present in the outer fibrous layer (OFL), the inner fibrous layer (IFL), and on chondrocytes in the cartilaginous layer (arrowheads). (C) Scleral section incubated with Mel1c receptor antibody followed by incubation in secondary antibody conjugated to green fluorescent dye. Mel1c labelling is most intense in the outer fibrous layer (OFL) and is also present on the inner fibrous layer (IFL) and on chondrocytes in the cartilaginous layer (arrowheads). D) Merged image of Mel1a and Mel1c labelled sclera. Yellow colour indicates areas of co- localization of Mel1a and Mel1c. Scale bar=100 μm.
Fig. 3.
Peptide block controls for Mel1a and Mel1c labelling in the Xenopus laevis cornea. (A and C) Corneal sections were incubated with green fluorescent dye conjugated Mel1a or Mel1c receptor antibodies previously pre-incubated with their corresponding peptide and were counterstained with DAPI blue nuclear dye. Absence of green stain indicates complete block of specific antibody binding with specific peptide. (B and D) Corneal sections were incubated with red fluorescent dye-conjugated Mel1a receptor antibody together with green fluorescent dye n conjugated Mel1c receptor antibody previously pre- incubated with either Mel1a or Mel1c peptide and counterstained with DAPI blue nuclear dye. Red labelling indicates areas of specific Mel1a receptor labelling in epithelium and green labelling indicates areas of specific Mel1c receptor labelling in surface epithelium, endothelium (arrowheads) and stroma. Scale bar=100 μm.
Fig. 4.
Peptide block controls for Mel1a and Mel1c labelling in the Xenopus laevis sclera. (A and C) Scleral sections were incubated with green fluorescent dye conjugated Mel1a or Mel1c receptor antibodies previously pre- incubated with their corresponding peptide and were counterstained with DAPI blue nuclear dye. Absence of green stain indicates complete block of specific antibody binding with specific peptide. (B and D) Scleral sections were incubated with red fluorescent dye-conjugated Mel1a receptor antibody together with green fluorescent dye conjugated Mel1c receptor antibody previously pre-incubated with either Mel1a or Mel1c peptide and counterstained with DAPI blue nuclear dye. Red labelling indicates areas of specific Mel1a receptor labelling in outer and inner fibrous layers (OFL and IFL; arrowheads) as well as in the cartilaginous layer and green labelling indicates areas of specific Mel1c receptor labelling in outer and inner fibrous layers (OFL and IFL; arrowheads) and in the cartilaginous layer. Scale bar=100 μm.
Fig. 5.
Confocal image of the differential distribution and co- localization of Mel1a and Mel1c receptor immunoreactivity in the Xenopus laevis cornea and sclera. Sections of Xenopus eyes were incubated first with the Mel1c receptor antibody and labelled with a green fluorescent dye-antibody conjugate then incubated with the Mel1a antibody labelled with a red fluorescent dye-antibody conjugate and analyzed by confocal microscopy. The merged image of Mel1c and Mel1a immunolabeling demonstrates the differential expression and co-localization of the two receptor types. (A) The two receptors appear to have some co- localization in the superficial layers of the corneal epithelium (small arrowheads), as indicated by the yellow fluorescence. Only the Mel1a red immunolabeling appears in the deeper layers of the corneal epithelium (large arrowheads). Some specific Mel1c green immunolabeling appears in corneal fibroblasts (small arrows) and in the corneal endothelium (large arrowheads), but the labelling that occurs in the non-cellular corneal stroma was determined to be artifact. In additional its expression in the corneal epithelium, Mel1a is present in the corneal stroma fibroblasts (small arrows) and the corneal endothelium (large arrowheads). (B) The two receptors are co- localized in the deep layers of the outer fibrous layer of the sclera (small arrowheads), as indicated by the yellow fluorescence. Mel1a and Mel1c are also co- localized to the inner fibrous layer of the sclera (small arrows). The matrix of the cartilaginous layer is not immunoreactive with the antibodies, whereas the chondrocytes (large arrows) display co- localization and/or differential expression of Mel1a and Mel1c. Scale bar=20 μm.
