Mertk triggers uptake of photoreceptor outer segments during phagocytosis by cultured retinal pigment epithelial cells

The RCS rat is a widely studied model of recessively inherited retinal degeneration. The genetic defect, known as rdy (retinal dystrophy), results in failure of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segment membranes. We previously used positional cloning and in vivo genetic complementation to demonstrate that Mertk is the gene for rdy. We have now used a rat primary RPE cell culture system to demonstrate that the RPE is the site of action of Mertk and to obtain functional evidence for a key role of Mertk in RPE phagocytosis. We found that Mertk protein is absent from RCS, but not wild-type, tissues and cultured RPE cells. Delivery of rat Mertk to cultured RCS RPE cells by means of a recombinant adenovirus restored the cells to complete phagocytic competency. Infected RCS RPE cells ingested exogenous outer segments to the same extent as wild-type RPE cells, but outer segment binding was unaffected. Mertk protein progressively co-localized with outer segment material during phagocytosis by primary RPE cells, and activated Mertk accumulated during the early stages of phagocytosis by RPE-J cells. We conclude that Mertk likely functions directly in the RPE phagocytic process as a signaling molecule triggering outer segment ingestion.     

Phagocytosis of lectin-coated beads by dystrophic and normal retinal pigment epithelium

In the dystrophic pigmented Royal College of Surgeons (RCS) rat, the retinal pigment epithelium (RPE) has a diminished capacity to phagocytose shed photoreceptor outer segments (ROS). An alteration in phagocytic recognition or ligand-receptor interactions between the RPE and ROS's could contribute to this defect. To this end, we have examined whether or not RPE lectin receptors are implicated in phagocytosis in the normal and dystrophic rat RPE by comparing differences in phagocytic uptake of lectin-coated beads. To test this, the following lectins were bound either indirectly to sugar-coated latex beads or directly to activated beads: Concanavalin A (conA), specific for mannose; Ulex europeus (ULEX), specific for fucose; Lens culinaris (LcH), specific for mannose; and wheat germ agglutinin (WGA), specific for N-acetyl glucosamine and sialic acid. The distribution of the lectin binding around beads was visualized and confirmed using lectin-Ferritin conjugates. Lectin-coated beads were fed to normal and dystrophic pigmented RPE tissue explants to determine differences in phagocytic uptake. We found that whether beads were directly or indirectly coated, similar results were obtained, but that there were differences in uptake of two types of lectin-coated beads by dystrophic as compared with normal animals. The dystrophic RPE phagocytosed greater numbers of conA-mannose beads (6.9/cell) than the normal RPE (3.6/cell). LcH-mannose beads were also phagocytosed by dystrophic (2.7/cell) but not by the normal (0/cell). A similar number of ULEX-fucose beads were taken up by dystrophic (3.8/cell) and normal (3.4/cell) RPE and neither took up WGA-N-acetyl glucosamine beads (0/cell). These results showing that the dystrophic RPE takes up greater numbers of conA and LcH-coated beads than the normal RPE suggest that a ligand-receptor interaction involving mannose may contribute to this difference in phagocytic uptake.     

Ultimate fate of rod outer segments in the retinal pigment epithelium.

To investigate the degradation pathway of rod outer segments (ROS) in vivo, we injected gold-labeled ROS into the subretinal space of rabbits using a pars plana approach. Histology and electron microscopy performed on the specimens 72 hr after ROS injection revealed that the retina over the injection site was reattached, the retinal pigment epithelial (RPE) cells were intact, and gold granules were localized inside melanin granules and melanosomes. These results indicate that, in RPE, in vivo degradation of ROS is associated with melanosomes.     

alpha2 But not alpha1 AMP-activated protein kinase mediates oxidative stress-induced inhibition of retinal pigment epithelium cell phagocytosis of photoreceptor outer segments

Oxidative stress causes retinal pigment epithelium (RPE) cell dysfunction and is a major risk factor leading to the development of dry-type age-related macular degeneration. Taking pharmacological and genetic approaches, we address the mechanisms by which sublethal oxidative stress inhibits RPE cell phagocytosis. Sublethal oxidative stress dose-dependently inhibited RPE cell phagocytosis of photoreceptor outer segments (POS) and activated AMP-activated protein kinase (AMPK) as determined by increased Thr172 and Ser79 phosphorylation of AMPKalpha and its substrate acetyl-CoA carboxylase, respectively. Similar to oxidative stress, 5-aminoimidazole-4-carboxamide riboside (AICAR), a pharmacological activator of AMPK, inhibited RPE cell phagocytosis of POS in a dose-dependent manner. Inhibition of RPE cell phagocytosis by AICAR was fully reversed by blockade of AICAR translocation into cells by dipyridamole or inhibition of AICAR conversion to ZMP by adenosine kinase inhibitor 5-iodotubercidin. In agreement, AICAR-induced activation of AMPK was abolished by preincubation with dipyridamole or 5-iodotubercidin. Knock-out experiments further revealed that alpha2 but not alpha1 AMPK was involved in RPE cell phagocytosis and that activation of alpha2 AMPK contributed to the inhibition of RPE cell phagocytosis by oxidative stress. Inhibition of RPE cell phagocytosis by activation of alpha2 AMPK was associated with a dramatic increase in acetyl-CoA carboxylase phosphorylation. In comparison, AMPK had no role in oxidative stress-induced breakdown of RPE barrier function. Taken together, reduction in POS load under oxidative stress might direct RPE cells to a self-protected status. Thus, activating AMPK could have therapeutic potential in treating dry macular degeneration.     

The effect of inhibitors of glycoprotein synthesis and processing on the phagocytosis of rod outer segments by cultured retinal pigment epithelial cells

Retinal pigment epithelial cells selectively phagocytize rod outer segments by a process that may be mediated by specific cell surface receptors. Since many receptors are glycoproteins, we have studied the effect of tunicamycin, an inhibitor of N-linked oligosaccharide synthesis, and of castanospermine and swainsonine, which are inhibitors of oligosaccharide processing, on the ability of cultured retinal pigment epithelial cells to phagocytize rod outer segment. Tunicamycin inhibits the glycosylation of newly synthesized glycoproteins by 85-90%; concomitantly, the phagocytosis of rod outer segments is inhibited by 70-80%. The effect of tunicamycin is to initially reduce rod outer segments binding, and therefore the subsequent ingestion of rod outer segments. SDS-PAGE analysis and autoradiography of [35S]methionine labelled extracts of tunicamycin-treated cells, demonstrates the disappearance of a number of glycoprotein bands, and the appearance of a number of protein bands of lower Mr. Kinetic analysis of the disappearance and reappearance of specific glycoproteins suggests that the lower Mr bands are the non-glycosylated forms of the higher Mr bands. By contrast, castanospermine and swainsonine have no effect on the ability of retinal pigment epithelial cells to phagocytize rod outer segments, or on the SDS-PAGE pattern of treated cells, although they were shown to inhibit oligosaccharide processing as expected. These results support the hypothesis that rod outer segment phagocytosis by retinal pigment epithelial cells is mediated by specific glycoprotein receptors. N-Glycosylation of these receptors is required for their function, or for their insertion into the plasma membrane, whereas processing of the N-linked oligosaccharide chains of these receptors is not crucial for rod outer segment phagocytosis by retinal pigment epithelial cells.     

Potassium currents in cultured cells of the rat retinal pigment epithelium

Whole-cell currents were investigated in cultured rat retinal pigment epithelial (RPE) cells. Two voltage-dependent conductances were discriminated. First, at potentials more positive than −30 mV, a time-dependent outward current was activated. Inhibition by Ba²⁺ (10 mM) and 4-aminopyridine (10 mM) indicated that this current was carried by potassium ions. This current showed no inactivation during 5 sec depolarizations. Second, an inward current, sensitive to Ba²⁺ (10 mM) and 4-aminopyridine (10 mM), was activated at potentials more negative than — 70 mV. Under extra- and intracellular potassium-free conditions, both currents disappeared. In summary, cultured rat RPE cells expressed one potassium conductance similar to the delayed rectifier and one similar to the inward rectifier. The delayed rectifier expressed characteristics comparable with those known in mammalian species and different from those in non-mammalian species.     

Combination of retinal pigment epithelium cell-conditioned medium and photoreceptor outer segments stimulate mesenchymal stem cell differentiation toward a functional retinal pigment epithelium cell phenotype

Recent studies have suggested that bone marrow-derived mesenchymal stem cells (BMMSCs) are capable of retinal tissue-specific differentiation but not retinal pigment epithelium (RPE) cell-specific differentiation. Photoreceptor outer segments (POS) contribute to RPE development and maturation. However, there has been no standard culture system that fosters the differentiation of BMMSCs into mature RPE cells in vitro. In this study, we investigated if the soluble factors from RPE cells and POS could differentiate BMMSCs into cells having a phenotype characteristic of RPE cells. Rat BMMSCs were separately co-cultured with RPE cells, or they were exposed to either control medium, RPE cell-conditioned medium (RPECM), POS, or a combination of RPECM and POS (RPECM-POS). After 7 days, the cells were analyzed for morphology and the expression of RPE markers (cytokeratin 8, CRALBP, and RPE65) to assess the RPE differentiation. Significantly higher pigment accumulation and increased protein expression of the three markers were seen in cells cultured in RPECM-POS than in other treated cultures. Furthermore, the RPECM-POS-treated cultures displayed ultrastructural features typical of RPE cells, expressed RPE cell functional proteins, and had the capability to phagocytose POS. Together, theses results suggest the combination of RPECM and POS stimulate BMMSCs differentiation toward a functional RPE phenotype. Our results provide the foundation for a new route to RPE regenerative therapy involving BMMSCs. Future work isolating the active agent in RPECM and POS would be useful in therapies for RPE diseases or in developing appropriately pre-differentiated BMMSCs for tissue-engineered RPE reconstruction.     

Endocytosis in the rat retinal pigment epithelium

Endocytosis in the retinal pigment epithelium (RPE) of rats was studied using horseradish peroxidase, microperoxidase and ferritin tracers. Tracer uptake was mediated by coated pits and coated vesicles. Coated pits formed at two discrete regions at the RPE plasma membrane: that portion of basal membrane directly opposing Bruch's membrane, and at the bases of the apical lamellae and villi. Two populations of coated vesicles were identified and distinguished by size, location and function. Large coated vesicles (91.8 +/- 14.7 nm in diameter) were located near the cell surface and incorporated tracer. Small coated vesicles (64.5 +/- 15.7 nm diameter) located more deeply within the cell were not tracer-labeled, and were often fused with the endoplasmic reticulum or the Golgi apparatus. Observations of the endocytic pathway in rat RPE cells are presented. Tracer was also found in organelles of the lysosomal system, e.g. the multivesicular body, but was not identified in the smooth endoplasmic reticulum or Golgi apparatus.     

Membrane turnover in rod photoreceptors: ensheathment and phagocytosis of outer segment distal tips by pseudopodia of the retinal pigment epithelium

We have documented the ultrastructural changes that occur within the photoreceptor outer segment and the retinal pigment epithelium (RPE) during photosensitive membrane turnover. We employed an in vitro eyecup preparation from Xenopus laevis in which a large shedding event was induced by adding the excitatory amino acid L-aspartate (Greenberger & Besharse 1985; J. comp. Neurol. 239, 361-372). We found that during L-aspartate-induced shedding the RPE cells formed, on their apical domains, previously undescribed processes that were directly involved in disc phagocytosis. These processes are structurally similar to processes formed by macrophages during phagocytosis and are accordingly referred to as pseudopodia. Pseudopodia were distinguishable from the apical villous process normally extended from the RPE in that they were closely applied to the surface of the outer segment, had a cytoplasmic matrix of low electron density that was devoid of most cellular organelles and were enriched in thin (7 nm diameter) filaments. Filament size, specific pseudopodial staining with the actin-specific probe rhodamine phalloidin and inhibition of pseudopod formation by cytochalasin D suggested that the thin filaments were composed of actin. Pseudopodial formation also occurs during a normal light-initiated shedding event. However, the low frequency of shedding, the asynchrony of the individual shedding events and the transient appearance of the pseudopodia prevented a full appreciation of their role during normal disc shedding. Associated with massive shedding and pseudopodial formation, there was an increased adherence between retina and RPE. During L-aspartate treatment, the apical portions of the RPE cells partitioned with the distal outer segment during retinal isolation. This effect was directly related to the development of pseudopodia and may reflect alteration of surface features of the rod outer segment (ROS)-RPE interface related to phagocytosis. Our observations show that transiently forming pseudopodia are the organelles of phagocytosis and that they may play a role in disc detachment as well.     

Ascorbate uptake in normal and diabetic rat retina and retinal pigment epithelium

Oxidative stress is an important causative factor in the pathogenesis of diabetic retinopathy. Therefore, it becomes important to understand the mechanisms that help maintain appropriate levels of a small molecule antioxidant such as ascorbate in the retina. The outer blood-barrier which results from the tight junctions between the retinal pigment epithelial cells (RPE) restricts the flow of nutrients reaching the retina. In this study, we characterized the transport properties of carboxyl-(14)C ascorbate (AA) in normal rat retina and RPE, and compared them with those in streptozotocin-diabetic rats. Retina and RPE accumulated AA by a temperature-sensitive and energy-dependent kinetic mechanism with an apparent K(M) of 380 and 420 microM, respectively. Accumulation of AA was significantly reduced in a sodium-free medium. Although high glucose concentrations reduced AA uptake by 40%, this was not affected by cytochalasin B. The RPE and retina of diabetic rats presented lower levels of AA accumulation. These findings suggest the presence of the specific vitamin C transporter SVCT in retina and RPE, which may be involved in the manifestation of diabetic retinopathy.     

A quantitative study of intramembrane changes during cell junctional breakdown in the dystrophic rat retinal pigment epithelium

Previous electron microscope freeze-fracture and tracer studies have revealed that intercellular junctions in the retinal pigment epithelium (RPE) of Royal College of Surgeons (RCS) rats with inherited retinal dystrophy [5] break down between three and six postnatal weeks [6, 7]. In this study quantitative computer techniques were used to analyze the freeze-fracture changes in the dystrophic RPE. The following parameters were measured: length of tight junctional strands/micron2; number of tight junctional strand anastomoses/micron2; number of gap junctional aggregates/micron2; area of gap junctional aggregates/micron2; and density of background intramembrane particles/micron2. At three postnatal weeks, the dystrophic junctional complex membrane is similar to normal, but at 10 weeks and later there are dramatic decreases in tight junctional strand length/micron2 and number of anastomoses/micron2, as well as in the number/micron2 and area of gap junctions/micron2, while the density of background particles/micron2 is dramatically increased. Correlational analysis revealed that changes in gap and tight junctions were significantly related to each other and to the increase in background particle density. The diameter of background particles within the normal and post-breakdown dystrophic junctions was measured in order to see whether the dispersal of gap and tight junctional particles (8-10 nm) into the surrounding membrane contributes to the increased particle density. These measures showed that background particles in all size ranges were more numerous in the dystrophic RPE, but that the largest increase was in the smallest diameter particles (6-7 nm). Thus, while gap and tight junctional sized particles contribute to the increase, particles from other sources may also be involved. Particle density of apical and basal membranes in the normal and in the 10 week and older dystrophic RPE was analyzed to study the effects of tight junctional breakdown on the distribution of intramembrane particles. These measures showed that particle density was greater basally than apically in the normal RPE and that particle density in both membranes decreased slightly in the dystrophic RPE, but that their ratio remained unchanged. It has been shown previously that even a single intact tight junctional strand is sufficient to maintain differences in particle density between apical and basal surfaces [14, 15] and in the majority of abnormal dystrophic junctional complexes at least one tight junctional strand remains intact.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of potassium transport in cultured retinal pigment epithelium and retinal glial cells by serum and epidermal growth factor.

The ionic environment of retinal photoreceptors is partially controlled by potassium transporters on retinal glial and retinal pigment epithelial cells (RPE). In this study, serum and epidermal growth factor (EGF) were examined as modulators of potassium transport in confluent cultures of human RPE and rabbit retinal glia. EGF is a known mitogen for confluent RPE cultures and was shown here to also stimulate [3H]thymidine incorporation in cultures of retinal glia. For potassium transport studies 86Rb was used as a tracer during a 17-min incubation. For both retinal cell types the mean total 86Rb uptake in 10% serum was approximately 60% above basal, serum-free controls. For EGF, tested in several experiments in a concentration range from 1 to 100 ng/ml, maximal total uptake was 33 and 24% above controls for RPE and glia, respectively. Inhibitor studies suggested that basal and serum-stimulated uptake for both cell types occurred by the ouabain-sensitive Na-K ATPase pump and by the furosemide- or bumetanide-sensitive Na-K-Cl cotransporter. EGF-stimulated uptake appeared to be due predominantly to the cotransporter. The data suggest that serum components and EGF, which may be available to retina-derived cells under pathologic conditions, may not only stimulate proliferation but may also act as short-term modulators of potassium ion movement and thus affect physiologic processes that are sensitive to ion homeostasis.     

Changes in retinal pigment epithelial cell autofluorescence and protein expression associated with phagocytosis of rod outer segments in vitro.

The accumulation of autofluorescent lipofuscin was quantified in cultured human retinal pigment epithelial (RPE) cells phagocytosing bovine rod outer segments (BROS) and the expression of proteins in these cells was investigated. Results showed a steady increase in autofluorescence of RPE cells over a 4-week period as measured by fluorophotometric flow cytometry. A significantly greater increase in autofluorescence was found in the cultured RPE cells from a 7-year-old donor compared with those from a 47-year-old donor. Within both groups the BROS-challenged cells had significantly higher fluorescence readings than the control cells which were not challenged. Autoradiography of 35S-labelled proteins separated by polyacrylamide gel electrophoresis (PAGE) revealed a small distinct band at 102 kDa in BROS-challenged RPE cells of both bovine and human origin that did not appear in control or microsphere-phagocytosing RPE cells. The intensity of the signal was unrelated to the duration of the challenge period.     

Acetyl- and butyrylcholinesterase molecular forms in normal and streptozotocin-diabetic rat retinal pigment epithelium.

We studied the composition of molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in normal and streptozotocin-induced diabetic rat retinal pigment epithelium (RPE). Tissues were sequentially extracted with saline (S(1)) and saline-detergent buffers (S(2)). About a 50% decrease in AChE molecular forms was observed in the diabetic RPE compared to the controls. Approximately 70% of the BChE activity in normal RPE was brought into solution and evenly distributed in S(1) and S(2). Analysis of the fractions from RPE revealed the presence of G(A)(1), G(A)(4) and a small proportion of G(H)(4) BChE forms in S(1); whereas G(A)(4) and G(A)(1) molecules predominate in S(2). A 40% decrease in the activity of G(A)(4) in S(2) was observed in the diabetic RPE. Our results show that diabetes caused a remarkable decrease in the activity of cholinesterases molecular forms in the RPE. This might be related to the alterations observed in diabetic retinopathy.     

Basement membrane stimulates the polarized distribution of integrins but not the Na,K-ATPase in the retinal pigment epithelium.

The basement membrane stimulates the differentiation and polarity of simple transporting epithelia. We demonstrated for the retinal pigment epithelium (RPE) of chicken embryos that polarity develops gradually. Although the RPE and an immature basement membrane are established on embryonic day 4 (E4), the distribution of the Na,K-ATPase and a family of basement membrane receptors containing the beta 1 subunit of integrin is nonpolarized. The percentage of polarized cells increases gradually until cells in all regions of the epithelium are polarized on E11. During this time, the basement membrane increases in size and complexity to form Bruch's membrane. To study the ability of the basement membrane to stimulate the polarized distribution of the beta 1 integrins or the Na,K-ATPase, RPE was harvested from E7, E9, or E14 embryos and cultured on Bruch's membrane isolated (in association with the choroid) from E14 embryos. As a control, the RPE was plated on the side of the choroid lacking a Bruch's membrane. The distribution of the beta 1 integrins and the Na,K-ATPase was determined by indirect immunofluorescence. Bruch's membrane stimulated the polarized distribution of the beta 1 integrins regardless of the developmental age of the RPE even though E7 RPE is nonpolarized in vivo. To examine the role of individual matrix components, RPE was plated on matrix-coated filters. The polarized distribution of the beta 1 integrins was stimulated by laminin, collagen IV, and Matrigel but not by fibronectin. Interestingly, laminin and collagen IV are present in the basement membrane on E4 when RPE is not polarized in vivo. Under no circumstances was the distribution of the Na,K-ATPase polarized. These data indicate that the basement membrane influences the distribution of a subset of plasma membrane proteins but that other factors are required for full polarity.     

Synthesis of retinoids by human retinal epithelium and transfer to rod outer segments.

In order to gain an insight into the pathogenesis of mouse muscular dystrophy, we investigated the natural suppressor serine tRNA. The natural suppressor seryl-tRNA was distinguished from the other seryl-tRNAs on the basis of its specific property of being converted into phosphoseryl-tRNA by a tRNA kinase. On a wet-weight basis, the content of total tRNA in dystrophic muscles was 47% of that in normal muscles. Although the serine-accepting activities of tRNA were similar in muscles of 3-month-old dystrophic and normal mice, the ratio of [32P]phosphoseryl-tRNA (suppressor tRNA) to the total serine tRNA was significantly enhanced in dystrophic muscles compared with that in normal muscles. This high content of suppressor tRNA in dystrophic muscles was further confirmed by dot-blot hybridization experiments with the DNA probes CGTAGTCGGCAGGAT and CGCCCGAAAGGTGGAA for major tRNA(IGASer) and suppressor tRNA respectively. At the early postnatal age of 3 weeks, when only a week had elapsed since the first manifestation of the dystrophic symptom (hindleg dragging), the ratio of suppressor tRNA to major tRNAs in dystrophic hindleg muscles was abnormally increased. Thereafter it decreased with age in normal mice but remained almost unchanged in dystrophic mice. Consequently, at 3 months old, it was 1.7 times higher in dystrophic than in normal mice. The suppressor tRNA is now accepted to play a role in the synthesis of glutathione peroxidase. The present study showed that the content of this enzyme was abnormally elevated in dystrophic mice. Previously we had demonstrated that the docosahexaenoic (C22:6) acid content in phospholipids was decreased, possibly resulting from the enhanced oxidative milieu caused by the dystrophic condition. Thus far, the findings suggest that an increase in the contents of suppressor tRNA and glutathione peroxidase in dystrophic muscle may have been secondarily induced by such a highly oxidative state in the dystrophic condition. However, it is difficult to exclude the possibility that the natural suppressor tRNA plays a primary role in the pathogenesis of muscular dystrophies.     

Light-induced oxidation of photoreceptor outer segment phospholipids generates ligands for CD36-mediated phagocytosis by retinal pigment epithelium: a potential mechanism for modulating outer segment phagocytosis under oxidant stress conditions

Clearance by the retinal pigment epithelium (RPE) of shed photoreceptor outer segments (OSs), a tissue with one of the highest turnover rates in the body, is critical to the maintenance and normal function of the retina. We hypothesized that there is a potential role for photo-oxidation in OS uptake by RPE via scavenger receptor-mediated recognition of structurally defined lipid peroxidation products. We now demonstrate that specific structurally defined oxidized species derived from arachidonyl, linoleoyl, and docosahexanoyl phosphatidylcholine may serve as endogenous ligands on OSs for uptake by RPE via the scavenger receptor CD36. Mass spectrometry studies of retinal lipids recovered from dark-adapted rats following physiological light exposure demonstrate in vivo formation of specific oxidized phosphatidylcholine molecular species possessing a CD36 recognition motif, an oxidatively truncated sn-2 acyl group with a terminal gamma-hydroxy(or oxo)-alpha,beta-unsaturated carbonyl. Cellular studies using RPE isolated from wild-type versus CD36 null mice suggest that CD36 plays a role in engulfment, but not initial binding, of OSs via these oxidized phospholipids. Parallel increases in OS protein-bound nitrotyrosine, a post-translational modification by nitric oxide (NO)-derived oxidants, were also observed, suggesting a possible role for light-induced generation of NO-derived oxidants in the initiation of OS lipid peroxidation. Collectively, these studies suggest that intense light exposure promotes "oxidative tagging" of photoreceptor outer segments with structurally defined choline glycerophospholipids that may serve as a physiological signal for CD36-mediated phagocytosis under oxidant stress conditions.     

Regeneration of retinal pigment epithelium damaged in rat foetuses by 6-aminonicotinamide

The pathological changes induced by 6-AN (administered i. p. on day 15 of pregnancy) in the pigment epithelium of the retina were studied on days 17, 18, 19 and 20 of pregnancy by light and electronmicroscopy. The marked vacuolar degeneration (enlargement of the perinuclear cisterns) observed until day 18 of pregnancy is followed on days 19 and 20 by a practically total regeneration, by the restitution of the normal microscopic and submicroscopic feature. Within the eye, the regenerative phenomena mentioned are limited to the pigment epithelium (although initially similar pathological changes are observed also in its other components.