Phagocytosis of Photoreceptor Outer Segments during Retinal Development In Utero A R T H U R W. S P I R A AND PETER T. HUANG Division of Morphological Science, The University of Calgary, Calgary, Alberta, Canada T2N 1Nr
ABSTRACT The retinal pigmented epithelium of the fetal guinea pig was examined for evidence of phagocytosis and degradation of outer segments. Beginning at the early stages of outer segment formation and continuing through the remaining fetal period, pigment epithelial cells contained large and small phagosomes with multi-lamellated internal structure. Thus, mammalian retinal photoreceptor cells which normally develop and mature in utero in the absence of light undergo a process of phagocytosis of outer segment fragments comparable to that found in species whose retinae mature postnatally outside of the uterus. The processes and factors involved in the normal renewal of photoreceptor outer segments in the retina of the mature animal is beginning to be clarified. Renewal occurs by the continued production of new outer segment membranes (Young, '67) accompanied by the phagocytosis of the apices of outer segments and incorporation as phagosomes into pigmented epithelial (PE) cells (Young and Bok, '69; Spitznas and Hogan, '70). Phagocytosis of outer segments occurs according t o a diurnal cycle of PE cell activity (LaVail, '76; Basinger et al., '76), with the peak of the cycle following soon after the onset of lighting. The role of light in regulating the cycle is uncertain. Amphibians kept in total darkness do not demonstrate the surge of phagocytosis expected a t the time lights are normally turned on (Basinger et al., '76; Hollyfield e t al., '76), but the rat exhibits periodic activity according t o a circadian pattern even after three days in the dark (LaVail, '76). Phagocytosis is also an integral element in establishing a balanced rate of outer segment growth (renewal and disposal) during development. In the postnatal mouse (LaVail, '73; Feeney, '73) and rat (Ishikawa and Yamada, '70; Kuwabara and Weidman, '74) phagosomes are present shortly after outer segments first develop. Whether light is required for the stimulation of phagocytic activity during development cannot be deduced from these studies. In altricial animals the photoreceptors mature postnatally, with light unAM.
J. ANAT. (1978)152: 523-628
doubtedly reaching the developing photoreceptor cells through the closed eyelids. The fetal guinea pig may be more suitable for studying the role of light versus intrinsic factors in outer segment development. Its outer segments develop in utero beginning in the seventh week of gestation, three weeks before birth (Spira, '75). The present study examines the pigment epithelium of the fetal guinea pig for evidence of outer segment phagocytosis and degradation. METHODS
Pregnant albino Hartley guinea pigs were kept in cycling light (12L: 12D). Fetuses aged 45 to 63 days (near-term) were exteriorized under halothane anesthesia. Following enucleation, eyes were fixed by periorbital perfusion with 5% glutarlaldehyde-2% paraformaldehyde in cacodylate buffer for ten minutes followed by immersion for two and one-half hours at 4°C. Sections were cut through the retina adjacent to the optic disc, stained with toluidine blue or methylene blue for light microscopy or uranyl acetate and lead citrate for electron microscopy. RESULTS
Light microscopy of the posterior retina indicated the presence of densely stained spherical or irregularly shaped bodies (fig. 1) in the pigment epithelial cells. Dense bodies Accepted January 3, '78. ' Supported by the Medical Research Council of Canada.
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were located in apical, middle and basal cell regions. The largest of these reached 3.0 pm in length. Electron microscopy confirmed that dense bodies of this size were membrane-bound multilamellated bodies (figs. 2-4),identifiable as phagosomes (Spitmas and Hogan, '70). The membranes within phagosomes were in various states of disarray. Phagosomes frequently were closely associated with smaller membrane-bound dense bodies, spherical or ovoid in shape and with a dense core surrounded by a thin halo (fig. 4). These presumably were lysosomes (Ishikawa and Yamada, '70). Small phagosomes, less than 3.0 pm in diameter, containing fewer lamellae of disc membranes, were also present in the fetal PE cells (fig. 5).
ment of outer segments (Hollyfield et al., '76). This nibbling away of outer segments is not altered by changing conditions of illumination. If activity occurs in the fetus comparable to that present in the mature animal, then continuous phagocytic activity may occur throughout t h e period of outer segment growth; superimposed on this activity a more massive ingestion of outer segment fragments may periodically be initiated by some unknown stimulus. ACKNOWLEDGMENTS
The authors wish to express their gratitude to Doctor J. P. Wyse for his valuable discussion of the problems in this field of study, and to Mrs. A. Kana for her technical assistance.
DISCUSSION
LITERATURE CITED
The frequency of occurrence of large phagosomes suggests that photoreceptor outer segments are shed in utero on a significant scale. The presence of putative lysosomes in the PE in close association with large phagosomes likely indicates that ingested outer segment fragments are degraded in utero, as occurs postnatally (Ishikawa and Yamada, '70). Unlike the phagocytic process which occurs in the retinae of other rodents postnatally, that in the fetal guinea pig is not likely to be directly affected by light. It is thus of interest to know whether, in the absence of stimulation by light, phagocytosis in utero occurs a t a constant rate over a 24-hour period, or whether it follows a circadian pattern of variation comparable to that present in the mother or one inimical to the fetus. Studies are underway to explore this problem. The significance of the small phagosomes seen in the fetal PE cells is uncertain. They have been regarded as representing the remains of degraded large phagosomes (Ishikawa and Yamada, '701, and alternately as evidence of the phagocytosis of small frag-
Basinger, S., R. Hoffman and M. Matthes 1976 Photoreceptor shedding is initiated by light in the frog retina. Science, 194: 1074-1076. Feeney, L. 1973 t h e interphotoreceptor space. I. Postnatal ontogeny in mice and rats. Devel. Biol., 32: 101-114. Hollyfield, J. G., J. C. Besharse and M. E. Rayborn 1976 The effect of light on the quantity of phagosomes in the pigment epithelium. Exp. Eye Res., 23: 623-635. Ishikawa, T., and Y. Yamada 1970 The degradation of the photoreceptor outer segment within the pigment epithelial cell of rat retina. J. Elect. Microscop., 19: 85-91. Kuwabara, T., andT. A. Weidman 1974 Development of t h e prenatal r a t retina. Invest. Ophthal., 13: 725-739. LaVail, M. M. 1973 Kinetics of rod outer segment renewal in t h e developing .~ mouse retina. J. Cell Biol., 58: 650-661. 1976 Rod outer segment disk shedding in rat retina: relationship t o cyclic lighting. Science, 194: 1071-1074. Spira, A. W. 1975 In utero development and maturation of the retina of a nonprimate mammal: a light and electron microscopic study of t h e guinea pig. Anat. Embryol., 146: 279-300. Spitznas, M., and M. 3. Hogan 1970 Outer segments of photoreceptors and the retinal pigment epithelium. Arch. Ophthal., 84: 810.819. Young, R. W. 1967 The renewal of photoreceptor cell outer segments. J. Cell Biol., 33: 61-72. Young, R. W., and D. Bok 1969 Participation of the retinal pigment epithelium in t h e rod outer segment renewal process. J. Cell Biol., 42: 392-403.
PLATE
Abbreviations ap, apical processes bi, basal infoldings BM, Bruch’s membrane C, choroid
L, lysosomes OS, outer segments p, phagosome PE, pigment epithelium
PLATE 1 EXPLANATlON OF FIGURES
1 Light micrograph of the photoreceptor cells and pigment epithelium in a near-term guinea pig fetus. The pigment epithelium contains densely stained phagosomes (arrows). x 960.
2 Pigmented epithelium of a near-term guinea pig fetus. Phagosomes are here located in mid-cell and adjacent to the basal infoldings. x 15,000. 3 A large phagosome in a fetal pigment epithelial cell contains a whorl of densely packed membranes. X 21,000. 4 Fetal pigment epithelial cell with two large phagosomes and lysosomes.
X
22,000.
5 Apical cytoplasm of a pigment epithelial cell and part of an adjacent outer segment in a 45-day-old fetal guinea pig. A small phagosome in t h e pigment epithelium contains several whorls of closely packed membranes in a dense matrix. x 60,000.
526
PHAGOCYTOSIS OF OUTER SEGMENTS IN FETAL RETINA Arthur W Spira and Peter T. Huang
PLATE I
ABSTRACT The retinal pigmented epithelium of the fetal guinea pig was examined for evidence of phagocytosis and degradation of outer segments. Beginning at the early stages of outer segment formation and continuing through the remaining fetal period, pigment epithelial cells contained large and small phagosomes with multi-lamellated internal structure. Thus, mammalian retinal photoreceptor cells which normally develop and mature in utero in the absence of light undergo a process of phagocytosis of outer segment fragments comparable to that found in species whose retinae mature postnatally outside of the uterus. The processes and factors involved in the normal renewal of photoreceptor outer segments in the retina of the mature animal is beginning to be clarified. Renewal occurs by the continued production of new outer segment membranes (Young, '67) accompanied by the phagocytosis of the apices of outer segments and incorporation as phagosomes into pigmented epithelial (PE) cells (Young and Bok, '69; Spitznas and Hogan, '70). Phagocytosis of outer segments occurs according t o a diurnal cycle of PE cell activity (LaVail, '76; Basinger et al., '76), with the peak of the cycle following soon after the onset of lighting. The role of light in regulating the cycle is uncertain. Amphibians kept in total darkness do not demonstrate the surge of phagocytosis expected a t the time lights are normally turned on (Basinger et al., '76; Hollyfield e t al., '76), but the rat exhibits periodic activity according t o a circadian pattern even after three days in the dark (LaVail, '76). Phagocytosis is also an integral element in establishing a balanced rate of outer segment growth (renewal and disposal) during development. In the postnatal mouse (LaVail, '73; Feeney, '73) and rat (Ishikawa and Yamada, '70; Kuwabara and Weidman, '74) phagosomes are present shortly after outer segments first develop. Whether light is required for the stimulation of phagocytic activity during development cannot be deduced from these studies. In altricial animals the photoreceptors mature postnatally, with light unAM.
J. ANAT. (1978)152: 523-628
doubtedly reaching the developing photoreceptor cells through the closed eyelids. The fetal guinea pig may be more suitable for studying the role of light versus intrinsic factors in outer segment development. Its outer segments develop in utero beginning in the seventh week of gestation, three weeks before birth (Spira, '75). The present study examines the pigment epithelium of the fetal guinea pig for evidence of outer segment phagocytosis and degradation. METHODS
Pregnant albino Hartley guinea pigs were kept in cycling light (12L: 12D). Fetuses aged 45 to 63 days (near-term) were exteriorized under halothane anesthesia. Following enucleation, eyes were fixed by periorbital perfusion with 5% glutarlaldehyde-2% paraformaldehyde in cacodylate buffer for ten minutes followed by immersion for two and one-half hours at 4°C. Sections were cut through the retina adjacent to the optic disc, stained with toluidine blue or methylene blue for light microscopy or uranyl acetate and lead citrate for electron microscopy. RESULTS
Light microscopy of the posterior retina indicated the presence of densely stained spherical or irregularly shaped bodies (fig. 1) in the pigment epithelial cells. Dense bodies Accepted January 3, '78. ' Supported by the Medical Research Council of Canada.
523
524
ARTHUR W. SPIRA AND PETER T. HUANG
were located in apical, middle and basal cell regions. The largest of these reached 3.0 pm in length. Electron microscopy confirmed that dense bodies of this size were membrane-bound multilamellated bodies (figs. 2-4),identifiable as phagosomes (Spitmas and Hogan, '70). The membranes within phagosomes were in various states of disarray. Phagosomes frequently were closely associated with smaller membrane-bound dense bodies, spherical or ovoid in shape and with a dense core surrounded by a thin halo (fig. 4). These presumably were lysosomes (Ishikawa and Yamada, '70). Small phagosomes, less than 3.0 pm in diameter, containing fewer lamellae of disc membranes, were also present in the fetal PE cells (fig. 5).
ment of outer segments (Hollyfield et al., '76). This nibbling away of outer segments is not altered by changing conditions of illumination. If activity occurs in the fetus comparable to that present in the mature animal, then continuous phagocytic activity may occur throughout t h e period of outer segment growth; superimposed on this activity a more massive ingestion of outer segment fragments may periodically be initiated by some unknown stimulus. ACKNOWLEDGMENTS
The authors wish to express their gratitude to Doctor J. P. Wyse for his valuable discussion of the problems in this field of study, and to Mrs. A. Kana for her technical assistance.
DISCUSSION
LITERATURE CITED
The frequency of occurrence of large phagosomes suggests that photoreceptor outer segments are shed in utero on a significant scale. The presence of putative lysosomes in the PE in close association with large phagosomes likely indicates that ingested outer segment fragments are degraded in utero, as occurs postnatally (Ishikawa and Yamada, '70). Unlike the phagocytic process which occurs in the retinae of other rodents postnatally, that in the fetal guinea pig is not likely to be directly affected by light. It is thus of interest to know whether, in the absence of stimulation by light, phagocytosis in utero occurs a t a constant rate over a 24-hour period, or whether it follows a circadian pattern of variation comparable to that present in the mother or one inimical to the fetus. Studies are underway to explore this problem. The significance of the small phagosomes seen in the fetal PE cells is uncertain. They have been regarded as representing the remains of degraded large phagosomes (Ishikawa and Yamada, '701, and alternately as evidence of the phagocytosis of small frag-
Basinger, S., R. Hoffman and M. Matthes 1976 Photoreceptor shedding is initiated by light in the frog retina. Science, 194: 1074-1076. Feeney, L. 1973 t h e interphotoreceptor space. I. Postnatal ontogeny in mice and rats. Devel. Biol., 32: 101-114. Hollyfield, J. G., J. C. Besharse and M. E. Rayborn 1976 The effect of light on the quantity of phagosomes in the pigment epithelium. Exp. Eye Res., 23: 623-635. Ishikawa, T., and Y. Yamada 1970 The degradation of the photoreceptor outer segment within the pigment epithelial cell of rat retina. J. Elect. Microscop., 19: 85-91. Kuwabara, T., andT. A. Weidman 1974 Development of t h e prenatal r a t retina. Invest. Ophthal., 13: 725-739. LaVail, M. M. 1973 Kinetics of rod outer segment renewal in t h e developing .~ mouse retina. J. Cell Biol., 58: 650-661. 1976 Rod outer segment disk shedding in rat retina: relationship t o cyclic lighting. Science, 194: 1071-1074. Spira, A. W. 1975 In utero development and maturation of the retina of a nonprimate mammal: a light and electron microscopic study of t h e guinea pig. Anat. Embryol., 146: 279-300. Spitznas, M., and M. 3. Hogan 1970 Outer segments of photoreceptors and the retinal pigment epithelium. Arch. Ophthal., 84: 810.819. Young, R. W. 1967 The renewal of photoreceptor cell outer segments. J. Cell Biol., 33: 61-72. Young, R. W., and D. Bok 1969 Participation of the retinal pigment epithelium in t h e rod outer segment renewal process. J. Cell Biol., 42: 392-403.
PLATE
Abbreviations ap, apical processes bi, basal infoldings BM, Bruch’s membrane C, choroid
L, lysosomes OS, outer segments p, phagosome PE, pigment epithelium
PLATE 1 EXPLANATlON OF FIGURES
1 Light micrograph of the photoreceptor cells and pigment epithelium in a near-term guinea pig fetus. The pigment epithelium contains densely stained phagosomes (arrows). x 960.
2 Pigmented epithelium of a near-term guinea pig fetus. Phagosomes are here located in mid-cell and adjacent to the basal infoldings. x 15,000. 3 A large phagosome in a fetal pigment epithelial cell contains a whorl of densely packed membranes. X 21,000. 4 Fetal pigment epithelial cell with two large phagosomes and lysosomes.
X
22,000.
5 Apical cytoplasm of a pigment epithelial cell and part of an adjacent outer segment in a 45-day-old fetal guinea pig. A small phagosome in t h e pigment epithelium contains several whorls of closely packed membranes in a dense matrix. x 60,000.
526
PHAGOCYTOSIS OF OUTER SEGMENTS IN FETAL RETINA Arthur W Spira and Peter T. Huang
PLATE I
