Pigmentation formation and expression analysis of tyrosinase in Siniperca chuatsi.

Animal pigmentation primarily depends on the presence and mixing ratio of chromatophores, functioning in animal survival and communication. For the benthic and carnivorous Siniperca chuatsi, pigmentation pattern is key to concealment and predation. In this study, the formation, distribution, and main pattern of chromatophores were observed in the embryos, larvae, skins, and visceral tissues from S. chuatsi. Melanophores were firstly visualized in the yolk sac at segmentation stage, and then they were migrated to the whole body and further clustered into the black stripes, bands, and patches. In adult S. chuatsi, the head, black band, and body side skins mainly contained melanophores, showing as deep or light black. The abdomen skin mainly contained iridophores, showing as silvery. In the eye, the pigment layers were located in the epithelial layers of iris and retina and shown as black. Then, the pigmentation-related gene, tyrosinase gene from S. chuatsi (Sc-tyr) was analyzed by bioinformatics and quantitative methods. The Sc-tyr gene encoded a protein with 540 amino acids (Sc-TYR). The Sc-TYR contained two copper ion binding sites, which were coordinated by six conserved histidines (H182, H205, H214, H366, H370, H393) and necessary for catalytic activity. The Sc-TYR was well conserved compared with TYR of various species with higher degree of sequence similarity with other fishes (77.6-98.3%). The qRT-PCR test showed that the Sc-tyr mRNA reached the peak value at segmentation stage in the embryo development, the black skins displayed a higher expression level than that in silvery skin, and the eye had the highest expression level compared with other tissues. Further research on enzyme activity showed that the expression patterns of tyrosinase activity were similar to that of the Sc-tyr mRNA. Comparing with the results of molecular and phenotype, it was found that the temporal and spatial distributions of tyrosinase corresponded well with changes in pigmentation patterns and the intensity of skin melanization. This study initially explored the pigmentation formation and tyrosinase expression, which served as a foundation for further insight into the genetics mechanism of body color formation in S. chuatsi.

The structure of anchovy outer retinae (Engraulididae, Clupeiformes) - a comparative light- and electron-microscopic study using museum-stored material.

The outer retinal architecture of Engraulididae is uncommon among vertebrates. In some anchovies, e.g., Anchoa, two cone types are arranged alternating in long photoreceptor chains, i.e., polycones. The cones have radially oriented outer segment lamellae in close contact with a complex guanine tapetum, most probably subserving polarization contrast vision. To clarify the distribution of the aberrant polycone architecture within the Engraulididae and to provide indications about polycone evolution, the outer retina morphology of 16 clupeoid species was investigated by light and electron microscopy, predominantly using museum-stored material. The outgroup representatives of four clupeid subfamilies (Clupeonella cultriventris, Dorosoma cepedianum, Ethmalosa fimbriata, Pellonula leonensis) show a row pattern of double cones, partially with single cones at defined positions and a pigment epithelium with lobopodial protrusions containing melanin. The pristigasterid Ilisha africana has double rows of single cones lying between linear curtains of pigment epithelium processes filled with minute crystallites and melanin concentrated near their vitreal tips. Within the Engraulididae, two main architectures are found: Coilia nasus and Thryssa setirostris have linear multiple cones or polycones separated by long pigment epithelium barriers containing tapetal crystallites and melanin in the tips (also found in Setipinna taty), whereas Anchoviella alleni, Encrasicholina heteroloba, Engraulis encrasicolus, Engraulis mordax, Lycengraulis batesii, and Stolephorus indicus exhibit the typical polycone architecture. Cetengraulis mysticetus and Lycothrissa crocodilus show cone patterns and pigment epithelium morphology differing from the other anchovy species. The sets of characters are compared, corroborated with the previous knowledge on clupeoid retinae and discussed in terms of functional morphology and visual ecology. A scenario on polycone evolution is developed that may serve as an aid for the reconstruction of engraulidid phylogeny. Furthermore, this study demonstrates the suitability of museum material for morphological studies, even at the electron microscopic level.

Redefining the limit of the outer retina in optical coherence tomography scans.

OBJECTIVE: A highly reflective layer seen in retinal optical coherence tomography (OCT) has been believed to correspond to the choriocapillaris (CHC) and retinal pigment epithelium (RPE). On gray-scale scans of OCT-2000, and on Stratus OCT, this layer by the outer retinal limit can be resolved into 2 distinct laminae. We analyzed these 2 laminae in normal and abnormal maculae to infer their anatomic correlate. DESIGN: Retrospective study. METHODS: Analysis of macular OCT scans was performed in 44 patients using OCT-2000, and in 39 patients using Stratus OCT. Thirty of these patients had no ocular disease, and their OCT was normal. The other 53 patients had several macular diseases of different etiologies. Both color and gray-scale images were analyzed. RESULTS: Macular OCT scans showed a double laminae at the level where the retina interfaces the RPE in normal subjects using both OCT-2000 and Stratus OCT. In 2-dimensional scans, this laminar structure appears as a double line. It is best distinguished on the Stratus OCT and gray-scale images of OCT-2000. This double line consisted of a thin inner line and a thicker outer line. Similar analysis in patients with macular pathology showed a discernible double line at the retina/RPE interface in at least part of the scan. However, in patients with macular hole, the area corresponding to the absent retina showed only a single line. The inner line component appeared to follow the contour of the retina. This phenomenon was also seen in eyes with neurosensory detachment secondary to central serous chorioretinopathy and other etiologies. In contrast, in macular pathologies where the outer retina did not lose contiguity with the RPE, such as in lamellar macular hole and in cystoid macular edema, the double line persisted. Software for retinal thickness measurements regularly place the outer limit of the retina at the internal aspect of the inner line, probably underestimating the retinal thickness by about 24 to 34 mum. CONCLUSIONS: A double laminar structure at the outer retina/RPE/CHC interface can be consistently distinguished on commercially available OCT of normal eyes. In eyes with macular pathology, OCT analysis of the inner lamina leads us to conclude it is most likely part of the neurosensory retina and not part of the RPE/CHC complex as previously thought.

Lack of type XVIII collagen results in anterior ocular defects.

Mice lacking type XVIII collagen have defects in the posterior part of the eye, including delayed regression of the hyaloid vasculature and poor outgrowth of the retinal vessels. We report here that these mice also have a fragile iris and develop atrophy of the ciliary body. The irises of Col18a1-/- mice can be seen to adhere to the lens and cornea. After the pupils begin to function, the double layer of epithelial cells separates at the apical cell contacts, leading to defoliation of its posterior pigment epithelial cell layer, and extracellular material begins to accumulate in the basement membrane zones of the iris. In contrast to the iris epithelia, where no clear signs of cellular atrophy were detected, the lack of type XVIII collagen resulted in atrophy of the pigmented epithelial cells of the ciliary body, and there were also ultrastructural abnormalities in the basement membrane zones. These changes did not lead to chronically elevated intraocular pressures, however. Our results indicate that type XVIII collagen is needed for the integrity of the epithelial basement membranes of the iris and the ciliary body and that its gene should therefore be taken into account as a new potential cause of anterior segment disorders in the eye.

Relation among serum and tissue concentrations of lutein and zeaxanthin and macular pigment density.

BACKGROUND: Lutein and zeaxanthin are the only carotenoids in the macular region of the retina (referred to as macular pigment [MP]). Foods that are rich in lutein and zeaxanthin can increase MP density. Response to dietary lutein and zeaxanthin in other tissues has not been studied. OBJECTIVE: The objective of this study was to examine tissue responses to dietary lutein and zeaxanthin and relations among tissues in lutein and zeaxanthin concentrations. DESIGN: Seven subjects consumed spinach and corn, which contain lutein and zeaxanthin, with their daily diets for 15 wk. At 0, 4, 8, and 15 wk and 2 mo after the study, serum, buccal mucosa cells, and adipose tissue were analyzed for carotenoids, and MP density was measured. RESULTS: Serum and buccal cell concentrations of lutein increased significantly from baseline during dietary modification. Serum zeaxanthin concentrations were greater than at baseline only at 4 wk, whereas buccal cell and adipose tissue concentrations of zeaxanthin did not change. Adipose tissue lutein concentrations peaked at 8 wk. Changes in adipose tissue lutein concentration were inversely related to the changes in MP density, suggesting an interaction between adipose tissue and retina in lutein metabolism. To investigate the possibility of tissue interactions, we examined cross-sectional relations among serum, tissue, and dietary lutein concentrations, anthropometric measures, and MP density in healthy adults. Significant negative correlations were found between adipose tissue lutein concentrations and MP for women, but a significant positive relation was found for men. CONCLUSION: Sex differences in lutein metabolism may be an important factor in tissue interactions and in determining MP density.

Localized depigmentation of the retinal pigment epithelium and macrophage invasion of the retina in the bullfrog.

An unusual condition of the inferior retinal pigment epithelium (RPE) and neural retina has been observed in essentially all the large bullfrogs examined (Rana catesbeiana, 13 to 20 cm body length, supplied from the U.S. West Coast and Midwest). By ophthalmoscopy the inferior fundus exhibited numerous white spots and lines, which were found by light microscopy to be overlain by smaller black dots and lines. Closer examination revealed that the light areas were regions of depigmented RPE and that the black dots and lines were melanosome-laden macrophages within the adjacent retina. Further examination by light microscopy and electron microscopy allowed the formulation of the following sequence. (1) Monocytes in the choroidal capillaries crossed Bruch's membrane and passed vitreally between adjacent RPE cells. (2) In the subretinal space monocytes transformed into phagocytic macrophages, which became engorged with melanin granules and other RPE inclusions, whereas nearby RPE cells became much thinner and very depigmented. (3) The pigment-laden macrophages then moved vitreally into the avascular neural retina. Although in most areas only the RPE appeared affected by macrophage invasion, occasional localized photoreceptor disruption occurred. The severity of the lesion varied with frog size, being pronounced in large frogs, moderate in medium-sized animals, and absent in small frogs. Because the pigmentary changes were localized to the inferior part of the eye (which receives the most light from the sun overhead) of large bullfrogs (which are likely old), this phenomenon may be due to a change in RPE melanin granules resulting from the cumulative effect of light exposure.

Improved use of tapetal reflection for eye-position monitoring.

A new technique is described for eye-position monitoring in species with strong tapetal reflections. A fiber optic is used to introduce light into the eye, whose optics then produce an image of the fundus on a tangent in front of the animal. The technique simplifies heretofore tedious measurement of cylotorsional changes, as well as providing a very wide view of the fundus. It has been used successfully in conjunction with single-unit recording from the visual system.

Acoustic microscopy of the human retina and pigment epithelium.

An acoustic microscope uses sound waves rather than light to image a sample, and displays viscoelastic rather than optical properties. The Stanford instrument, operating at frequencies near 1,000 MHz, achieves resolution and magnification that is comparable to a light microscope. Using this instrument, we examined sections of normal human retina and pigment epithelium and found that characteristic degrees of acoustic attenuation or phase shift were produced by structures such as cell nuclei, rod and cone outer segments, Bruch's membrane, red blood cells, and ocular pigment. Resolution was better with thin than thick sections, and fixation did not significantly alter the acoustic properties of the tissues studied. A comparison of iris tissue from albino and pigmented rabbits showed that melanin was a particularly strong acoustic attenuator. Acoustic microscopy may provide a new and direct means of probing the physical structure of tissues and cells.

Ocular pigmentation in white and Siamese cats.

Ocular pigmentation in white cats with blue and yellow eyes and in Siamese cats was examined ophthalmoscopically and histologically. Yellow-eyed white cats had entirely normal ocular pigmentation. Blue eyes of white cats had normal pigmentation of the iridial and retinal pigment epithelia but no stromal pigmentation of the iris or choroid. This deficit is apparently due to the absence of stromal pigment cells, certainly in the iris. As a general rule, the blue eye of white cats had no tapetum. Siamese cats had reduced pigmentation of the iridial and retinal pigment epithelia and no stromal pigmentation of the iris or choroid. The lack of pigmentation is apparently due to the inability of stromal pigment cells to produce pigment, certainly in the iris. We conclude that the abnormality of visual pathways previously described in the Siamese cat is not due simply to a deficiency of pigment of cells of neural crest origin.

Retinal pigment epithelial cells of the posterior pole have fewer Na/K adenosine triphosphatase pumps than peripheral cells.

The density of Na/K adenosine triphosphatase (ATPase) pumps in retinal pigment epithelial (RPE) cells in different retinal regions was quantified by measuring the binding of 3H-ouabain to RPE in cow and human eyecups. In bovine eyes, pump density was estimated in RPE samples isolated from three retinal regions outlined with a 7-mm trephine: one from the posterior pole in the area centralis and two from the superior, equatorial retina representing unpigmented (in the tapetum) and pigmented zones. In human eyes, RPE samples were isolated from a posterior region centered around the macula and one superior region. Ouabain binding to RPE of the posterior pole of both species was approximately 40-60% lower than binding to RPE of more peripheral regions in the same eyes. For bovine eyes, ouabain binding did not differ between pigmented and unpigmented cells of the superior retina, suggesting that reduced binding in the relatively amelanotic posterior cells was not related to levels of pigmentation. For human RPE, binding to posterior cells was lower in eyes from donors of all ages (range, 17-90 yr). The data suggest that Na/K ATPase pump site density is lower in posterior RPE cells of both bovine and human eyes, perhaps due to a regional difference in requirements for ionic regulation.

Interphotoreceptor matrix domains ensheath vertebrate cone photoreceptor cells.

The retinal interphotoreceptor matrix (IPM) occupies the space between the neural retina and the retinal pigmented epithelium (RPE), two neuroectoderm-derived epithelia. While the IPM appears to be a major route by which photoreceptor cells receive vital metabolic factors, relatively little is known concerning its structure and function. The studies reported here describe the presence of specialized domains of the IPM that ensheath cone, but not rod, inner and outer segments in pig, monkey, and human retinae. These cone extracellular matrix sheaths are chemically and structurally distinct from the remainder of the IPM as revealed by their specific binding of the lectin peanut agglutinin (PNA) and their structural stability during physical dissociation of the retina. Biochemical studies suggest that the PNA-binding components of the cone matrix sheaths are trypsin-sensitive glycoproteins. These structures may play a role in establishing a specialized microenvironment for cone photoreceptors, maintaining proper orientation of cone outer segments, and/or facilitating cone-RPE interactions.

Retinal pigment epithelial lipofuscin and melanin and choroidal melanin in human eyes.

Optical measurements of the pigments of the retinal pigment epithelium (RPE) and choroid were made on 38 human autopsy eyes of both blacks and whites, varying in age between 2 wk and 90 yr old. Lipofuscin in melanin-bleached RPE was measured as fluorescence at 470 mm following excitation at 365 nm and was found to be proportional to fluorescence measured at 560 nm in unbleached tissue. Transmission measurements of RPE and choroidal melanin were converted and expressed as optical density units. The choroidal melanin content increased from the periphery to the posterior pole. RPE melanin concentration decreased from the periphery to the posterior pole with an increase in the macula. Conversely, the amount of RPE lipofuscin increased from the periphery to the posterior pole with a consistent dip at the fovea. There was an inverse relationship between RPE lipofuscin concentration and RPE melanin concentration. The RPE melanin content was similar between whites and blacks. Lipofuscin concentration was significantly greater (P = 0.002) in the RPE of whites compared to blacks; whereas blacks had a significantly greater (P = 0.005) choroidal melanin content than whites. The amounts of both choroidal and RPE melanin showed a trend of decreasing content with aging, whereas the amount RPE lipofuscin tended to increase (whites greater than blacks). Per fundus area, the amount of choroidal melanin was always greater than that in the RPE. There was a statistically significant (P = 0.001) increase in RPE height with age, most marked in eyes of whites after age 50 and correlated with the increase in lipofuscin concentration.

Retinomotor pigment migration in the teleost retinal pigment epithelium. I. Roles for actin and microtubules in pigment granule transport and cone movement.

In lower vertebrates, retinal pigment epithelial (RPE) cells and photoreceptors undergo dramatic "retinomotor movements" in response to changes in light conditions. In the dark, RPE pigment granules aggregate to the (choroidal) base of the RPE cells, cones elongate, and rods contract. In the light, movements are reversed: pigment granules migrate out into the long apical projections of the RPE cells, cones contract, and rods elongate. In this report the time courses of dark-induced pigment aggregation and light-induced dispersion have been characterized (and compared to cone movements) in the blue stripe grunt, Haemulon sciurus. It was found that aggregation and dispersion occur at linear rates of 3.4-3.5 microns/min and that RPE movements are kinetically independent from cone movements induced by the same changes in light conditions. The roles of actin and microtubules in RPE and cone movements were also investigated by using the actin-inhibitors, cytochalasins-B and -D, and the microtubule inhibitor, colchicine. Light-induced pigment dispersion, as well as maintenance of the fully dispersed (light-adapted) position appear to require actin-dependent processes. Intraocularly injected cytochalasins-B and -D fully prevented pigment dispersion when administered to dark-adapted animals immediately prior to their exposure to light, and caused pigment aggregation to the RPE cell base when administered to fully light-adapted animals. Ultrastructural studies showed that actin filaments, which in untreated retinas were found closely associated with pigment granules and the plasma membrane, were disrupted after cytochalasin-B treatment. Both dispersive and aggregative pigment movements within the cell body appeared to require microtubule-dependent processes. Intraocularly injected colchicine disrupted microtubules and blocked pigment granule translocation in both directions in the cell body. A hypothetical model to explain pigment movements in response to changes in light conditions is proposed based on these observations as well as on data from the literature.

Distribution of melanosomes across the retinal pigment epithelium of a hooded rat: implications for light damage.

Distribution of melanosomes across the retinal pigment epithelium of hooded rats (Long-Evans) is studied at the light microscopic and electron microscopic levels. This distribution is shown to be nonuniform: more melanosomes exist in the periphery than elsewhere and, importantly, there are very few melanosomes in a restricted area of the central portion of the superior hemisphere compared with the corresponding part of the inferior hemisphere. The region with fewest melanosomes is precisely the one that is highly susceptible to light damage. Because this region is the same in both pigmented and albino eyes, the paucity of melanin in this region is not the cause of its great sensitivity to light damage. Nor does light cause the nonuniform distribution of melanin. A possible explanation, involving a proposed vestigial tapetum, is given in order to explain the correlation of melanosome counts and sensitivity to light damage.

Retinomotor pigment migration in the teleost retinal pigment epithelium. II. Cyclic-3',5'-adenosine monophosphate induction of dark-adaptive movement in vitro.

The retinal pigment epithelium (RPE) and photoreceptors of teleosts exhibit dramatic examples of cell motility (called retinomotor movements) in response to diurnal changes in lighting conditions. In darkness the pigment granules of the RPE migrate to the scleral base of the RPE cell and cone photoreceptors elongate. In the light these movements are reversed; pigment granules disperse into the long apical projections of the RPE cell and cones contract. It is reported here that treatments that elevate cytoplasmic cyclic AMP induce dark-adaptive movements (pigment aggregation and cone elongation) in light-adapted retinas cultured in the light. Treatments designed to elevate cGMP had no effect. In dose-response studies with the cAMP analog, dibutyryl cyclic AMP (dbcAMP), we found that the RPE pigment did not exhibit intermediate states of aggregation with increasing concentrations of dbcAMP but instead changed abruptly from the fully light-adapted to the fully dark-adapted retinomotor positions between 10 microM and 50 microM exogenous dbcAMP concentrations. Cones, on the other hand, elongated to intermediate extents in proportion to increasing dbcAMP concentration between 10 microM and 500 microM. These observations suggest that cytoplasmic cAMP plays a role in regulating retinomotor position in both RPE and cones.

The topography and age relationship of lipofuscin concentration in the retinal pigment epithelium.

Lipofuscin pigment granules (LPG) have been implicated as a marker of cellular aging. We have quantitated the content of LPG in human retinal pigment epithelium (RPE) as a function of age. Furthermore, topographic distribution of LPG within individual eyes was measured. Microspectrofluorometric determination of the distribution of LPG in human RPE cells revealed a progressive accumulation of LPG with increasing age. LPG first appeared in the basilar portions of RPE cells of young eyes. In older eyes, LPG formed into clumps and were noted to fill the entire RPE cell. The RPE topographic distribution of LPG revealed an increased accumulation in the posterior pole, with a consistent dip at the fovea. The ratio of lipofusion accumulation at the posterior pole, to the total RPE, remained constant throughout life.

Do blue-eyed white cats have normal or abnormal retinofugal pathways?

Three white cats that had blue eyes and no tapetum were studied by behavioral, electrophysiological, and anatomical methods in order to determine whether they showed evidence of abnormal retinofugal pathways comparable to those found in Siamese cats and in other mammalian forms having melanin deficits. The three cats were normal in every respect. However, several other white cats, obtained subsequently, do show an abnormality of the retinogeniculate pathway identical to the abnormality of Siamese cats. Cats of the second type are thought to be homozygous for the Siamese gene and also the express the White gene. Because the characteristics Siamese pigmented "points" fail to develop in the presence of the white gene, cats of the second type are not distinguishable from other white cats on the basis of eye color or coat color. In terms of their central visual pathways and of their patterns, however, they are recognizably Siamese. It is not known how common "crypto-Siamese" cats are in the white cat population, but the possibility of their occurrence suggests that, in general, white cats should not be used for studied of the central visual pathways.

The relationships of age changes in retinal pigment epithelium and Bruch's membrane.

PURPOSE: To study the correlations between age, Bruch's membrane (BM) thickness, retinal pigment epithelial (RPE) autofluorescence, and RPE residual body content. METHODS: Eight-millimeter-diameter macular discs from 88 unpaired human eye bank eyes were obtained within 72 hours of death, fixed in 10% neutral buffered formalin, and hemisected horizontally. One portion of the macular disc was embedded in paraffin and stained with periodic acid-Schiff for the measurement of BM thickness. RPE autofluorescence measurements were performed on unstained, deparaffinized sections. A second portion of the macular disc was prepared for electron microscopy to evaluate RPE residual body content. Linear and polynomial regression techniques were used to investigate the correlations between age, BM thickness, RPE autofluorescence, and RPE residual body content. RESULTS: Bruch's membrane thickness increased with age according to the linear model. RPE autofluorescence and RPE residual body content also increased with age, but the correlations were best approximated by a quadratic model. The correlations between RPE autofluorescence and residual body content and between BM thickness and RPE autofluorescence were best approximated by a linear regression model. There was considerable variation in these correlations between specimens and within the same age group. CONCLUSIONS: Although the changes in RPE and Bruch's membrane increased with age and there was a direct correlation between changes in the two tissues, there was considerable variation within each age group and between specimens. This probably reflects the multifactorial nature of the process.

Similarity of mRNA phenotypes of morphologically normal macular and peripheral retinal pigment epithelial cells in older human eyes.

PURPOSE: To determine the expression profiles of morphologically normal human retinal pigment epithelial (RPE) cells that originate from the macula and periphery. METHODS: Morphologically normal RPE cells from 15 human globes from donors aged 52 to 82 years old were laser capture microdissected. Total RNA from 5000 cells was SMART amplified, [33]P-labeled, and hybridized to a cDNA array containing 4325 known genes. Expression profiles were analyzed by hierarchical cluster analysis, Prediction Analysis of Microarrays (PAM), and Significance Analysis for Microarrays (SAM). Differentially expressed genes were evaluated further by real time RT-PCR. RESULTS: The overall expression profiles of RPE cells from the macula and periphery were similar. Unsupervised and supervised hierarchical cluster analysis showed that patient genotype was a stronger separating factor than topographical location. SAM analysis identified 11 genes that were underexpressed by macular RPE cells. The expression patterns of these 11 genes were confirmed by real time RT-PCR, with 5 genes reaching statistical significance. CONCLUSIONS: Whereas the overall expression profiles were similar between cells from the macula and periphery, subtle differential expression of five genes could contribute to RPE phenotypic differences based on topographic location.

Myeloid body associations in the frog pigment epithelium.

Myeloid bodies are found in the retinal pigment epithelium of certain vertebrate species. They are organized structural forms of the smooth endoplasmic reticulum which are usually seen as stacks of flattened, smooth saccules having a circular or lens-shaped configuration. Our findings in the frog Rana pipiens suggest that changes occur in the structure of the myeloid bodies which are related to the phase of the diurnal lighting cycle. At certain times, the myeloid bodies are found closely associated with other cytoplasmic organelles, notably the nucleus and oil droplet. In addition these associations can be induced by incubation of the isolated eyecup in the presence of guanosine 3',5'-monophosphate.