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Frog pineal photoreceptor renewalPreliminary observations.

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Frog Pineal Photoreceptor Renewal :
Preliminary Observations
Department of Biological Structure, Uriiversity of Washington,
SeutUe, Washington
The intracranial epiphysis of the adult frog, Rana pipiens, has
been examined by electron microscopic cytochemistry and radioautography. Acid
phosphatase is localized within vacuoles of macrophages free in the lumen, in
subspherical vesicles of the ellipsoid portions of photoreceptive cell inner segments, and in occasional heterogeneous cytoplasmic inclusions of the supportive cells.
The distribution of radioactivity in the epiphysis at intervals of 1, 3, 5, 9, 14,
25, and 60 days following injection of tritiated leucine, as determined by quantitative radioautography, is consistent with an hypothesized process of continual
renewal of photoreceptive cell outer segments. The pattern of radioautographic
labeling of the pineal photoreceptors, which resembles more closely that of retinal
cones than rods, is correlated with previous morphologic and electrophysiological
studies of these cells.
The radioautographic and cytochemical data suggest that macrophages within
the epiphyseal lumen are involved in phagocytosis and, ultimately, in digestion
of degenerate outer segments. They may perform a function similar to that of
pigment epithelial cells of the lateral eye retina.
Previous fine structural studies have
demonstrated that sensory cells comparable to the photoreceptors of the lateral eye
retina exist in the saccular pineal organs
of most nonmammalian vertebrates (see
Wurtman et al., '68). Whereas the outer
segments of these photoreceptive cells appear to be regularly structured in the lizard
parietal eye (Eakin and Westfall, '60;
Oksche and Kirschstein, '68), it has been
observed that in the intracranial epiphysis
of many species the outer segments are
often disarranged and appear degenerate,
even though the surrounding tissue is well
preserved (cyclostomes: Eakin, '63; Bertolini and Mangia, '66; cartilagenous and
bony fishes: Rudeberg, '68a,b, '69; Ueck,
'69; anzphibians: Eakin and Westfall, '61;
Kelly, '62, '65; Eakin et al., '63; Oksche and
von Harnack, '63; Oksche and Vaupel-von
Harnack, '63, '65; Kelly and Smith, '64;
Ueck, '68: Charlton, '68; Flight, '68; Hendrickson and Kelly, '71; reptiles: Eakin and
Westfall, '60; Collin, '67, '68; Wartenberg
and Baumgarten, '68; Oksche and Kirschstein, '68; Vivien-Roels, '69; and birds:
ANAT. REC., 171: 99-116.
Oksche and Vaupel-von Harnack, '65; Collin, '66; Oksche, '68; Quay et d., '68;
Bischoff, '69; Oksche and Kirschstein, '69;
Oksche et al., '69; Ralph, '70).
Since profiles resembling immature
stages in the development of outer segments were observed in the pineal of Rana
pipiens, i t was suggested (Kelly and Smith,
'64; Ueck, '68) that these photoreceptors
might be engaged in continual renewal of
outer segment components, as originally
postulated by Holmgren ('18) and demonstrated recently for photoreceptors of the
frog lateral eye retina (Young, '67, '68,
'69; Young and Droz, '68; Young and Bok,
'69, '70; Hall et al., '69; Bargoot et d.,
'69). This hypothesis was strengthened by
the observation of macrophages in the
pineal lumen, which were postulated
(Kelly and Smith, '64) to function in
phagocytosis and digestion of degenerate
outer segments.
Received Oct. 20, '70. Accepted Feb. 3, '71.
1 Present address: Department of Biological Structure, University of Miami School of Medicine, Miami,
Florida 33152.
distilled water, was applied to the grids
(Caro and Van Tubergen, ’62). Following
exposure in the dark at 10°C under low
humidity for eight weeks, the preparations
were developed in Microdol-X for five minutes at 18°C. rinsed in distilled water, and
fixed in Kodak Rapid Fix for five minutes.
The grids were stained with uranyl acetate
and lead citrate and examined in an RCA
2C 3r 3G electron microscope. Electron
micrographs were obtained of structures
surrounding the pineal lumen at a magniMATERIALS AND METHODS
fication of Y 7,000. Prints were prepared
Seven adult frogs ( R a m pipiens) aver- at a final enlargement of X 17,500. The
aging 25 gm body weight were each in- radioautographs were quantitated by dejected with 1 mc of L-le~cine-4,5-~H
(speci- termination of the number of grains per
fic activity 5.0 c/mmole, New England unit area of various cellular regions (table
Nuclear Corp., Boston, Mass.) without anes- 1 ) at each time period, using the Chalkley
thesia by way of the dorsal lymph sac. The procedure (Chalkley et al., ’49) as modifrogs were maintained at 20°C under con- fied by Ross and Benditt (’65). A total of
ditions of ordinary laboratory illumination 1122 grains was counted over labeled porand the epiphysis was removed from in- tions of 334 prints examined. Each print
dividual light-adapted animals at intervals depicted a separate area, and collectively
of 1, 3, 5, 9, 14, 25, and 60 days. The they represented samplings from all seven
tissue was fixed initially for two hours at animals.
20°C in a solution of 4% methanol-free
For cytochemical studies, pineal organs
formaldehyde and 1% sucrose in 0.1 M from adult, light-adapted frogs were fixed
phosphate buffer, washed overnight in 7% initially in a mixture of 1.8% glutaraldesucrose in 0.1 M phosphate buffer at 10°C. hyde and 0.5% sucrose in 0.1 M cacodylpostfixed for one hour in ice cold 1% ate buffer at 20°C for two hours, followed
osmium tetroxide in 0.1 M phosphate buf- by an overnight wash in 7% sucrose in
fer, dehydrated in an ethanol series, and the same buffer. Sections were cut at 100
embedded in Epon-812. Sections approxi- micrometers (Smith and Farquhar, ’66)
mately 1000 A thick of longitudinally and processed for the demonstration of
oriented pineals were cut with a diamond acid phosphatase by the method of Barka
knife on a Porter-Blum MT-2 ultramicro- and Anderson (’62). Control media were
tome and mounted on 200 mesh grids pre- prepared with omission of the substrate,
viously coated with parlodion and carbon. p-glycerophosphate or addition of 0.01 M
The grids were then coated with a thin sodium fluoride. The tissue was postfixed
layer of evaporated carbon and mounted in cacodylate buffered 1% osmium tetroxon glass microscope slides by means of ide, stained “en bloc” with 0.5% uranyl
tape. Ilford L-4 emulsion, diluted 1:2 with acetate in Michaelis buffer (Smith and
To test this hypothesis, the intracranial
pineal organ (epiphysis cerebri) of the
adult frog, Rann pipiens, has been studied
with cytochemical and radioautographic
techniques. Evidence will be presented in
support of the postulated process of renewal and casting off of outer segment
components by the pineal photoreceptors,
followed by uptake and probable degradation of this material by macrophages
within the lumen.
Grains per unit area following exposure of f r o g pineal to H3-Zeucine
Photoreceptor myoid
Photoreceptor ellipsoid
Photoreceptor outer segmc:nt
Photoreceptor nucleus
Lumen miscellaneous
Supportive cell nucleus
Supportive cell nucleus
Macrophage cytoplasm
Macrophage nucleus
Farquhar, '66), and further processed for
electron microscopy as outlined above.
For morphologic studies, pineal organs
were fixed according to the glutaraldehyde
and osmium procedure above or with ice
cold 3.75% osmium tetroxide in 0.05 M
s-collidine buffer for one hour, followed by
processing as above for electron microsCOPY.
the primary aldehyde fixation employed
here. This fixative also yields good preseivation of other components which are often
disrupted with primary osmium fkation,
including luminal macrophages and tubules of smooth endoplasmic reticulum in
the supportive cells.
Photoreceptive cells
As in the lateral eye retina (Nilsson,
'64), the photoreceptors of the pineal
organs of Rana pipiens consist of an outer
and inner segment which lie apical to the
nucleus and the synaptic body (fig. 1 ). The
inner segment is subdivided into the ellipsoid portion, comprised of numerous mitochondria, and the myoid portion, containing mainly rough endoplasmic reticulum
and Golgi complexes. The ellipsoid region
also exhibits subspherical, membranebounded vesicles containing spherical inclusions (approximately 60 nm in diameter) in a granular matrix of moderate
electron density (fig. 2).
Outer segments of the photoreceptors of
frog pineal organs resemble those of cones
of the lateral retina in size and in that the
membranous stacks usually remain in continuity with the plasma membrane (Eakin
and Westfall, '61 ), As described previously
(Kelly and Smith, '64), pineal outer segments often appear disorganized (fig. 2 )
in contrast to the regular organization OP
cone outer segments in the lateral eye
retina. Although sensitivity of outer segments to artif actual vesiculation following
osmium fixation is well known (Eakin,
'65; Rohlich, '66), the irregular images of
pineal outer segments persist even with
Among the outer segments of the photoreceptive cells within the lumen of the frog
epiphysis are free cells resembling the
macrophages reported in a variety of other
tissues (see Pearsall and Weiser, '70).
These cells are irregular in shape, frequently with pseudopodia (fig. 6) and contain numerous membrane-bounded inclusions. These inclusions may be classified
into three main types, although intermediate forms are common:
1. Myelin figures (figs. 5, 7, 12, 14),
some of which closely resemble outer segments of the photoreceptive cells (fig. 6).
2. Smaller vacuoles containing granular material of moderate electron density
(figs. 5, 6, 13).
3. Dense bodies (fig. 6 ) which are
often somewhat irregular in shape and
composed of a finely granular, electrondense material within which whorls of
membranous material may be embedded.
The macrophage cytoplasm is also characterized by occasional lipid droplets (fig.
5) and numerous fine filaments averaging
50 A in diameter (figs. 5, 6). These Baments are similar to those found in macrophages of Ambystoma and implicated in
motility and cytoplasmic streaming (Monroy, '70).
Supportive cells
cells are found among the
photoreceptive elements and are characGeneral cytologic features
terized by numerous profiles of smooth
The epiphysis of the adult frog is a sac- endoplasmic reticulum which appear to be
cular organ containing at least three cell continuous with myeloid bodies (fiq. 3 ) .
types : photoreceptors, supportive cells, and Although the smooth endoplasmic reticuluminal macrophages (Kelly and Smith, lum may appear vesiculated following pri'64). Ganglion cells, as noted in the pineal mary osmium fixation, with glutaraldehyde
organs of the anurans Rana esculenta fixation the cisternae are usually tubular.
(Oksche and Vaupel-von Harnack, '63) ; Membrane-bounded inclusions containing
Xenopzis, Hymenochirus, and Bombina granular material and myelin figures are
(Ueck, '68), were not observed in the pres- occasionally observed in the supportive
cells (fig. 4).
ent study.
The enzyme acid phosphatase is demonstrable cytochemically in three sites within
the frog epiphysis : luminal macrophages,
subspherical vesicles of the photoreceptor
ellipsoid, and cytoplasmic inclusions of the
supportive cells.
In the macrophages, electron dense reaction product is localized in numerous
vacuoles (fig. 7) which correspond in size
and shape to the vacuoles of moderate electron density illustrated in figures 5 and 13.
The limiting membranes of these vacuoles
are usually obscured by the dense reaction
product. Relatively little reaction product is
found in association with the large myelin
figures or the dense bodies.
In the photoreceptors, reaction product
is limited to the membrane-bounded subspherical vesicles of the ellipsoid (fig. s),
although in a single thin section, not every
vesicle within a given ellipsoid displays
reaction product.
In the supportive cells, reaction product
is localized within the heterogeneous cytoplasmic inclusions, which also contain
dense bodies and presumed lipid droplets
(fig. 9).
The myoid and ellipsoid portions of the
inner segments are heavily labeled at day
1 and show progressive loss of radioactivity
thereafter (table 1 and fig. 15). The outer
segments show a moderately high content
of label by day 1 (fig. 10) which declines
gradually through day 60. The initial labeling of the outer segments is not confined
to a descrete band but rather is found
throughout the membranous lamellae in a
manner similar to that described for cones
of the lateral eye retina by Young ('69).
The macrophage cytoplasm shows a
relatively high initial level of labeling
which is distributed throughout the cytoplasm and declines to very low levels by
day 25 and then increases again between
days 25 and 60 (table 1). At day 60, the
grains are localized mainly over vacuoles
containing membranous whorls (fig. 14).
Most labeling of the supportive cell cytoplasm occurs at day 3 (figs. 11, 12, 13),
followed by a gradual decline until the
period between days 14 and 25, when there
is a slight increase in the level of labeling.
Somewhat surprisingly, localization of
grains over the heterogeneous inclusions of
the supportive cells (figs. 4, 9) was not
observed at any time.
associated radioactivity
within the pineal lumen shows a progressive decline from the initial moderate level
(table 1 ) . Since the tissue was fixed with
paraformaldehyde. these grains aver the
luminal areas of the sections are presumably due to protein-bound label rather
than free amino acids (Peters and Ashley,
'67; Bergeron and Droz, '68).
Nuclei of the photoreceptor, supportive
and macrophage cells show moderate levels
of radioactivity at day 1, which decline
progressively through day 60.
The cytochemical demonstration of acid
phosphatase is frequently used as a marker
for lysosomal structures (Smith and
Farquhar, '66). Macrophages in a variety
of other tissues have been found to degrade
ingested material by means of acid hydrolases (Cohn et al., '66; Dumont, '69; Friend
et al., '69), and cells of the retinal pigment
epithelium, which function in the uptake
and digestion of degenerate photoreceptor
outer segments, are also rich in acid phosphatase-positive vesicles (Ishikawa and
Yamada, '70). In the present study, the
demonstration of abundant lysosomes in
luminal macrophages is consistent with
their hypothesized role in the digestion of
outer segment material, perhaps among
other materials.
The acid phosphatase reaction observed
in the subspherical vesicles of the photoreceptor ellipsoids identifies these structures
as probable lysosomes, similar to the multivesicular bodies noted in other cell types
(Smith and Farquhar, '66). The previous
study of Kelly and Smith ('64) revealed
that these vesicles were negative to a number of staining techniques at the light
microscopic level, but the test for acid
phosphatase was not employed. Lysosomes
have also been demonstrated in the
"pseudosensory cells" of the reptilian
epiphysis by the acid phosphatase reaction
(Vivien-Roels, '69) but those cells do not
contain the curious spherical inclusions
noted in the lysosomes of the photoreceptors here.
The pattern of labeling of the pineal
outer segments is similar to that observed
in cones of the frog lateral eye retina by
Young ('69) in that the initial grains over
the outer segments are not localized within
a band, but rather are found throughout
the membranous stacks. This finding reenforces the morphologic (Eakin and
Westfall, '61; Kelly and Smith, '64) and
electrophysiologic (Dodt and Morita, '64)
observations that the pineal photoreceptors
resemble cones more closely than rods of
the lateral eye retina.
The myoid portion of the inner sgement,
which is rich in rough endoplasmic reticulum and Golgi complexes, is heavily labeled
at day 1, probably reflecting initial incorporation of the radioactive amino acid into
protein. The ellipsoid, which is comprised
of closely packed mitochondria, also shows
maximum labeling at day 1, with progressive loss thereafter. This labeling pattern
may reflect movement of newly synthesized
protein from the myoid through the ellipsoid portion to the outer segment, as well as
possible protein synthesis by the mitochondria themselves (Bergeron and Droz,
'69). Specific labeling of the centriolar
region of the eilipsoid was not observed.
The initial general labeling of the macrophage cytoplasm may reflect incorporation
of the labeled amino acid into structural or
other proteins by the macrophages, with a
gradual decline in this process as the availability of labeled precursors within the
lumen decreases. The increase in labeling
of the macrophage cytoplasm between days
25 and 60 may reflect ingestion at this
time of previously labeled outer segment
material as it is cast off by the photoreceptors. This possibility is supported by the
presence of membranous material, similar
to that of outer segments of photoreceptors,
within vacuoles of the macrophages (fig.
6 ) and by the fact that grains are localized over vacuoles containing membranous whorls at day 60. The presence of
acid phosphatase within numerous vacuoles of the macrophages is consistent with
ultimate degradation of the phagocytosed
outer segment material by lysosomal
The slight increase in labeling of supportive cell cytoplasm by days 25 and 60
could indicate uptake of labeled outer segment material by the supportive cells, although specific localization of grains over
presumptive phagocytic vacuoles containing membranous material has not been observed. Indeed, very few of these vacuoles
have been noted in the present study and
little evidence of any kind suggesting
phagocytosis of degenerating outer segments by supportive cells has been
It should be noted that in a study of this
nature, in which the adequacy of sampling
is limited by the accessibility of the organ
studied and techniques employed, the data
should be regarded as preliminary and conclusions drawn from them as tentative.
Much more quantitative radioautography
is required for adequate resolution of the
dynamic cellular relationships within the
amphibian epiphysis.
However, the data from this study do
provide the first positive experimental
evidence, in any animal, to substantiate
the well-known hypothesis (Holmgren, '18;
Kelly and Smith, '64; Ueck, '68) that pineal
photoreceptors of the lower vertebrates are
engaged in continual renewal of outer segment components. The radioautographic
and cytochemical data further suggest that
the function of phagocytosis and ultimate
digestion of the cast-off outer segments in
the frog is performed primarily by luminal
macrophages. There is no unequivocal
evidence to suggest phagocytosis by the
supportive cells. In this respect, the epiphysis of the frog resembles that of the dogfish (Rudeberg, '68b. '69), anuran Hymenochirus (Ueck, ' 6 8 ) , lizard (Collin,
'67), and bird embryo (Collin, '66), and
differs from the pineal of the lamprey
(Bertolini and Mangia, '66), lungfish
(Ueck, '69), the anurans Xenopus and
Bombina (Ueck, '68), and newt (Kelly,
'65; Flight, '68; Hendrickson and Kelly,
'71, in which the supportive cells appear
likely to perform a major phagocytic role.
In view of recent studies by Young and
co-workers (Young, '67, '68, '69; Young
and Droz, '68; Young and Bok, '69, '70),
it is of interest to note that the pineal
photoreceptors resemble cones of the retina
in that radioautographic labeling of their
outer segments is not restricted to a disCrete band. However, the pineal photoreceptors
the rods Of the lateral
eye retina with respect to the time period
of outer segment protein turnover (Young
and Bok, ’69; Hall et al., ’69) and in that
ultimate degradation of outer segment material cast off from both photoreceptive
cells is accomplished by a separate phagocytic cell type, namely the luminal macrophage in the pineal and the pigment epithelial cell in the retina.
The authors are grateful to Drs. A.
Hendrickson, K. L. Wood, and D. Luchtel
for their critical review of the manuscript.
Supported by USPHS grants GM-136,
HE-2698 (for general support of electron
microscope facilities), and Postdoctoral
Fellowship GM-29732, all from the National Institutes of Health, and by NSF
grant GB-14098 to Dr. Kelly.
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1969 A difference between rods and
Macrophage. Lea & Febiger, Philadelphia, pp.
cones in the renewal of outer segment protein.
Invest. Ophthalmol., 8: 222-231.
Peters, T., Jr., and C. A. Ashley 1967 A n arte- Young, R. W., and D. Bok 1969 Participation
fact i n radioautography due to binding of free
of the retinal pigment epithelium in the rod
amino acids to tissues by fixatives. J. Cell Biol.,
outer segment renewal process. J. Cell Biol., 42:
33: 53-60.
1970 Autoradiographic studies on the
Quay, W. B., A. Renzoni and R. M. Eakin 1968
metabolism of the retinal pigment epithelium.
Pineal ultrastructure i n Melopsittacus undulaInvest. Ophthalmol., 9: 524-536.
tus with particular regard to cell types and
Young, R. W., and 13. Droz 1968 The renewal
functions. Riv. Biol., 61: 371-393.
of protein in retinal rods and cones. J. Cell
Ralph, C. L. 1970 Structure and alleged funcBiol., 39: 169-184.
tions of avian pineals. Am. Zool., 10: 217-235.
1 Diagrammatic representation of a photoreceptor of the adult frog
epiphysis. OS, outer segment; e, ellipsoid; m, myoid; n, nucleus, sb,
synaptic body.
Ellipsoid and outer segment ( 0 s ) portions of a pineal photoreceptor
illustrating the subspherical vesicles ( S). Note spherical inclusions
(arrows) within the granular matrix of the vesicles. M, mitochondria;
B, basal body. Osmium-collidine fixation. x 14,000.
Supportive cell of the frog epiphysis, illustrating the myeloid bodies
( m ) i n continuity with profiles of smooth endoplasmic reticulum
( SER) and the nuclear envelope (arrows). Glutaraldehyde-osmiumcacodylate fixation. x 16,000.
Cytoplasm of a supportive cell containing a myeloid body ( m ) and
presumed phagocytic vacuoles (P). Glutaraldehyde-osmium-cacodylate
fixation. x 22,000.
Ann H. Bunt and Douglas E. Kelly
A macrophage within the lumen of the pineal. Note filaments ( f )
and presumed lipid droplet (L). Vacuoles with contents of moderate
electron density (V) and membranous whorls ( M ) are also illustrated.
O S , outer segment. Glutaraldehyde-osmium-cacodylate fixation.
X 7,000.
A luminal macrophage containing a dense body ( D ) an6 nearly intact
outer segment (0s). Note the cytoplasmic filaments ( f ) , centriole
( C ) and pseudopodia (P ). Glutaraldehyde-osmium-cacodylate
X 7,000.
Ann H. Bunt and Douglas E. Kelly
Figs. 7, 8 and 9 illustrate cells of the pineal following incubation for
the localization of acid phospbatase. Glutaraldehyde-osmium-cacodylate
Within a Iuminal macrophage, electron-dense reaction product is
found i n membrane-bounded vacuoles (arrows) which correspond in
size to the vacuoles with contents of moderate electron density illustrated in figures 5 and 6. N, nucleus; M, myelin figure within a large
vacuole. x 7,000.
In the inner segment of a pineal photoreceptor, reaction product i s
found within a subspherical vesicle (arrows). OS, outer segment,
>< 9,700.
9 In pineal supportive cells, reaction product (arrows) is found within
a heterogeneous cytoplasmic inclusion which d S 0 contains dense
bodies I d ) and presumed lipid droplets (1). N, supportive cell nucleus.
Ann H. Bunt and Douglas E. Kelly
Figs. 10 through 16 illustrate electron microscope autoradiographs of
frog epiphysis at varying intervals following injection of tritiated leucine.
Paraformaldehyde-osmium-phosphate fixation.
10 One day post injection. Note grains (circled in white) over outer
segments ( 0 s ) of the photoreceprors. E, ellipsoid; S, supportive cell
cytoplasm; L, lumen. x 8.600.
11 Three days post injection. Note grains over outer segments ( 0 s ) :
ellipsoids ( E ) and supportive cell cytoplasm (S). The ellipsoids,
comprised of closely packed mitochondria, are easily confused a t low
magnification with the membranous stacks of the outer segments.
x 8,600.
Ann H. Bunt and Douglas E. Kelly
Three davs Post iniection. Note grains over outer segments ( 0 s )
and ellipsoids ( E ) but the absence of grains over the macrophage
( M ) . x 8,600.
Three days post injection. Note presence of grains over photoreceptor
outer segments ( 0 s ) and low labeling of macrophage cytoplasm ( M ) .
x 8,600.
14 Sixty days post injection. Grains are heavily localized over myelin
figures within vacuoles of a luminal macrophage. x 17,500.
.-.- ...-
Photoreceptor El lipsoid
Photoreceptor Myoid
Photoreceptor Outer Segment
Macrophage Cytoplasm
Lumen t m i x .
' 1 1 1
135 9 14
~. exDosure of frog pineol to H3-leucine
Fig. 15 Graphic representation of the distribution of silver grains per unit area over different
components in the frog pineal following exposure
to H2-leucine, as determined by electron microscopic autoradiography.
Ann H. Bunt and Douglas E. Kelly
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observations, pineal, renewalpreliminary, photoreceptor, frog
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