Diurnal change and prolonged dark effect on myeloid bodies in the retinal pigment epithelium of the leopard frog

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Abstract
Myeloid bodies (MBs) are multi-lamellar membrane structures which are widely distributed in the retinal pigment epithelium (RPE) of lower vertebrates. The functional significance of these organelles is not known, but they are believed to be associated with lipid processing in the retina. To determine the nature of the daily changes which occur in MBs, and to ascertain the best sampling time for MB isolation as a prelude to the biochemical characterization of these organelles, this study has assessed the occurrence and area density of MBs in the leopard frog (Ram pipiens) over a daily light–dark cycle. Eye tissues from light-entrained (12L:12D) frogs were sampled at 1, 4, 7, 10 hr after both lights-on and lights-off (n = 5). In addition, to determine whether MB formation is a circadian or light-driven event, the effect of a period of prolonged darkness on MB formation was also examined by maintaining frogs in the dark for an additional 1, 4, 7, and 10 hr at the end of a normal dark phase (n = 5). The number and area density as a percentage of total RPE cell area (area%) for phagosomes (PGs), oil droplets (ODs) and MBs were determined morphometrically using light and electron microscopy. Data were analyzed by a nested analysis of variance. Results indicated that: 1) PG numbers were elevated significantly following lights-on and were reduced almost to nil in the late dark period. With extended light deprivation, during the time of normal lights-on, phagosome number remained low. These results agree with the previous reports on photoreceptor outer segment shedding and phagocytosis in the frog and confirm the light-driven nature of this process in this animal; 2) neither OD numbers nor their area density showed any significant change over a normal light-dark cycle; 3) there is a significant change in MB area as a percentage of total RPE cell area (F = 51.04) over a normal light—dark cycle, being lower in the light and higher in the dark. However, this increase in MB area density is not due to a change in total MB numbers, but rather it reflects a shift in the population size of MBs from predominantly small organelles in the light to larger MBs in the dark. Finally, MBs showed no change in area density during a period of prolonged darkness when compared with the normal dark period of a diurnal cycle, suggesting that daily MB area density changes do not reflect a circadian event, but rather represent a light-driven phenomenon, similar to outer-segment shedding in the frog. In conclusion, there is a significant diurnal change in frog MB area density over a 24-hr period, with the best sampling time for MB isolation being late in the dark phase of a light-dark cycle.