Cell Tissue Res (1995) 282:363-366

Cell&Tissue
Research
9 Springer-Verlag 1995

Cells of the photoreceptor line in the pineal organ

of an adult marsupial, Didelphis albiventris
Mdnica M. del C. Gonzfilez*, Jorge M. Affanni

Instituto de Neurociencia (INEUCI), Consejo Nacional de Investigaciones Cientfficas y Tdcnicas (CONICET), Departamento de Ciencias
Biol6gicas, Facultad de Ciencias Exactas y Natnrales, Universidad de Buenos Aires, Argentina
Received: 20 March 1995 / Accepted: 23 June 1995

Abstract. We report the presence of atypical pinealocytes as components of epiphyseal follicles in the adult
South American opossum Didelphis albiventris. Their
main characteristic is a bulbous-shaped apical cytoplasmic extension which protrudes towards the follicular lumen among the microvilli and cilia of neighbouring
ependymal cells. They resemble the photoreceptor-like
pinealocytes of sauropsids and developing photoreceptors in the retina of newborn mammals. Morphological
characteristics enable us to classify them as cells of the
receptor line.
Key words: Pineal organ - Pinealocytes - Secretory rudimentary photoreceptors - Ultrastructure - Didelphis
aIbiventris (Marsupialia)

from pineal photoreceptors, of lower vertebrates (cf.

Collin and Oksche 1981). This view was supported by
Korf et al. (1986) who reported the presence of S-antigen in mammalian pinealocytes, which is a recognized
marker of photoreceptor cells. Recently, Tamotsu et al.
(1990) demonstrated that typical and modified or rudimentary photoreceptors coexist with typical (mammalian-like) pinealocytes in the pineal organ of the ancient
vertebrate Lampetra japonica. According to these authors this means that the mammalian pineal gland is the
result of gradual regression and final disappearance of
the typical and modified photoreceptors, and the progressive development of the typical pinealocytes (see
also Korf 1994).
The aim of this paper is to report the presence of cells
of the photoreceptor line which resemble rudimentary
photoreceptors in the pineal gland of an adult mammal.

Introduction
Materials and methods

Throughout vertebrate phylogeny, the pineal organ has

evolved from a sensoneuroendocrine structure (lower
vertebrates) to a purely endocrine one (mammals) (Collin 1968b, Collin 1969, 1971; Collin and Oksche 1981;
Oksche 1965). Secretory rudimentary photoreceptor
cells were described in sauropsids as intermediate or
transient cell forms of this evolutionary process. These
cells consist of a cellular body, an internal segment and
a modified outer segment, whose membranous disks appear irregular or even rudimentary (Collin 1966, 1967a,
1967b, 1967c, 1967d, 1968a, 1968b, 1969, 1971; Collin
and Oksche 1981; Korf 1994; Oksche 1968, 1971,
1972; Oksche and Kirschstein 1969; Oksche etal.
1969). This implies that the mammalian neuroendocrine
pinealocytes were developed by gradual transformation
This work was supported by the Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET)
* Present address and address for correspondence: Laboratoire de
M~dicine Exp6rimentale, Universit6 Claude Bernard, 8 Av.
Rockefeller, F-69008 Lyon, France

Six adult D. albiventris (3 males and 3 females; 3-4 kg b.w),

maintained under laboratory conditions, were studied. The animals were housed in individual cages with free access to Purina
dog chow and water, and submitted to a natural light-dark cycle.
Around midday and under white light, they were anesthetized
with sodium thiopental (60 mg/kg, i.p.) and perfused through the
heart with modified saline (NaC1 0.8%, saccharose 0.8%, glucose
0.4%), followed by a solution of 5% glutarald~hyde and 6% paraformaldehyde in 0.I M cacodylate buffer (pH 7.2). The perfusion
was performed with a vessel 1.20 m above the heart level, at a
speed of 20 ml/min during 40 min. The epithalamic region was
exposed and the pineal gland was immediately removed with a
very sharp razor blade under a stereoscopic microscope. Extreme
care was taken not to exert pressure on the pineal gland. It was
fixed in the same fixative for 2 h at room temperature. The time
lag between the end of the perfusion and the removal of pineal
gland was 5-7 min. After being washed in buffer, the tissues were
immersed in 2% osmium tetroxide at room temperature for 2 h.
They were left overnight in 2% uranyl acetate at 4 ~ C, dehydrated
in acetone and infiltrated with and embedded in Epoxy resin
(Epon 812). Transverse sections of the whole pineal gland were
stained with toluidine blue and observed under the light microscope to identify the zone of the follicles. Transverse ultrathin

Fig. 1A-D. Maie adult D. aIbiventris sacrificed in autumn, A, B

Longitudinal section through the secretory rudimentary photoreceptor. Schematic representation (A) and microphotograph (B) in
which the following structures can be identified: a) an apical cytoplasmatic extension (AP) protruding towards the follicular lumen
(L) among the microvilli (my), which shows a polarity of its cytoplasmic components: a distal zone (dis) with abundant elongated
mitochondria (m), numerous clear vesicles (v), microtubules (mt),
and a basal portion (has) with numerous lipofuscin granules (gr);
b) a neck (N) containing microtubules (mr) and establishing contacts with the neighboring cells (CC) via desmosomes (de); c) a

sut~ranuclear zone (SN) rich in organelles. In a small number of

atypical pinealocytes a cytoplasmic "cilium-like" extension was
observed (X). xll000. Bar." 1 Jam. C, D Sections at the level of
the follicular lumen (L). C Cross-section through the apical
cytoplasmic extension (AP); note concentric membranous arrangement (mb); nerve fiber (f); microvilli (my). M2000. Bar: 1 pm. D
Cross-section through an outer segment containing electron-dense
bodies (RES), and displaying cilia with a 9+0 and 8+1 pattern of
microtubules (arrowheads); note microvilli (my). x40000. Bar: 0.5
pin

365
sections (50-60 per pineal) of the follicular zone were double
stained in uranyl acetate and lead citrate. They were examined in a
transmission electron microscope, Siemens Elmiskop I.

Results and discussion

The general features of the pineal region are similar to

those described by Mc Nulty and Hazlett (1980) in Didelphis virginiana.
Atypical pinealocytes were detected in the caudal part
of the pineal gland of adult D. albiventris; they are a
component of the epiphyseal follicles of this region. We
consider these cells atypical because of their bulbous or
elongated cytoplasmic extension protruding into the follicular lumen among the microvilli and cilia of neighboring ependymat cells (Fig. 1A, B). In general, the basal
part of these processes displays lipofuscin granules
which are frequently vesicular with membranous arrangements at their periphery; the distal part and the cytoplasm close to the origin of the apical process show a
large number of elongated or round mitochondria of vesicular appearance, clear vesicles, which are seen isolated or forming groups, and microtubules (Fig. 1A, B).
The diameter of such cytoplasmic extensions decreases at the level of the apical border of the neighboring
columnar cells surrounding the luminal space (ependymal cell type). Numerous microtubules arranged parallel
to the longitudinal axis were observed in this narrow
"neck" region. The apical pinealocyte processes contact
the adjacent columnar cells via desmosomes (Fig. 1A,
B).
In a few of the atypical pinealocytes we observed that
the apical bulbous cytoplasmic process tapers along the
follicular lumen as an intermediate cytoplasmic "ciliumlike" extension. This structure shows microtubules along
its axis, few elongated mitochondria, electron-dense
bodies and clear vesicles (Fig. 1A, B).
Microtubules of cilia arranged in a 9+0 or 8+1 pattern, whorls of membranous arrangements and clear vesicles delimited by membranes were observed in the follicular lumen among the numerous microvilli and few
cilia of the neighbouring columnar cells (Fig. 1C, D).
Our observations show that the atypical pinealocytes of
the adult marsupial, Didelphis albiventris, resemble cells
of the photoreceptor line. Both share the following characteristics:
1) An apical cytoplasmic protrusion similar: a) to the
inner segment of the secretory rudimentary photoreceptors described in sauropsids (Collin 1966, 1967b, 1967c,
1967d, 1968a, 1969, 1971; Collin and Oksche 1981;
Oksche 1968, 1971, 1972; Oksche and Kirschstein 1969;
Oksche et al. 1969), b) to the inner segment of the developing photoreceptors in the retina of four-day-old rats
(Zimmerman and Tso 1975), and c) to the apical cytoplasmic process of pinealocytes in rats younger than 17
days old (Zimmerman and Tso 1975).
2) A "neck" with the same characteristics as those described in photoreceptors and pinealocytes of newborn
rats (Zimmerman and Tso 1975) and in secretory rudimentary photoreceptors in the epiphysis cerebri of

sauropsids (Collin 1966, 1967a, 1967b, 1967c, 1967d,

1968a, 1969, 1971; Collin and Oksche 1981).
3) The presence of an outer segment in the follicular
lumen displaying: a) an arrangement of microtubules of
the 9+0 or 8+1 type, which is characteristic of sensory
cilia, similar to those seen in the outer segment of rudimentary photoreceptors of the pineal gland of sauropsids
(Collin 1969, 1971; Collin and Oksche 1981; Oksche
1971, 1972; Oksche and Kirschstein 1969; Oksche et al.
1969) and to those in the retina of newborn rats (Zimmerman and Tso 1975); b) clear vesicles surrounded by
a membrane and membrane whorls of different thickness, already described in the pineal gland and retina of
newborn rats (Zimmerman and Tso 1975) and in the pineal of the lower vertebrates (Collin 1966, 1967a, 1969;
Collin and Oksche 1981; Oksche 1968, 1971, 1972;
Oksche and Kirschstein 1969; Oksche et al. 1969). Such
structures were interpreted by the above authors as traces of lamellar disks belonging to the outer segments of
photoreceptor-like cells.
These findings suggest that in DideIphis albiventris
the pinealocytes have not completely lost characteristics
of photoreceptor-like cells. It is in agreement with Korf
et al. (1986) who observed that pinealocytes of the opossum display photoreceptor-specific markers such as retinal S-antigen.
If we consider that our studies were performed in
adult marsupials, the atypical pinealocytes constitute a
very remarkable feature. As far as we know, such structures were never described in adult mammals. In other
adult mammals ontogeny leads to typical pinealocytes in
which only "photoreceptor-specific" proteins remain
(Korf et al. 1986, Korf 1994), According to the characteristics described above and following the criterion as
defined by Collin and Oksche (1981), we assume that
these cells belong to the receptor line, i.e. represent rudimentary photoreceptor elements. This study is the first
step to elucidate the functional significance of these rudimentary photoreceptors. It is open to discussion
whether they possess any sensory function.

Acknowledgements. The authors wish to thank Dr. Amanda Pellegrino de Iraldi and Professor Andreas Oksche for their advice and
critical reading of this paper, Professor Jean-Pierre Collin for his
discussion, and Mrs. Margarita Lopez for the microphotographs
obtained. The technical assistance of Mrs. Isabel Farias in the
preparation of ultrathin sections is gratefully acknowledged.
References

Collin JP (1966) Contribution ~t l'6tude des follicules de l'6piphyse embryonnaire d'oiseau. C R Acad Sci (Paris), S6r D
262:2263-2266
Collin JP (1967a) Structure, nature sdcr6toire, d6generescence
partieile des photordcepteurs rudimentaires 6piphysaires chez
Lacerta viridis (Laurenti). C R Acad Sci (Paris), S6r D
264:647-650
Collin JP (1967b) Le photordcepteur rudimentaire de l'6piphyse
d'oiseau: le prolongement basal chez le passereau Pica pica.
C R Acad Sci (Paris), Sdr D 265:48-51
Collin JP (1967c) Nouvelles remarques sur l'6piphyse de quelques
lacertilians et oiseaux. C R Acad Sci (Paris), S6r D
265:1725-1728

366
Collin JP (1967d) Rcchcrches prdliminaires sur los propri6t6s histochimiques de l"@iphyse de quelques lacertilien.s. C R Acad
Sci (Paris), S6r D 265:1827-1830
Collin JP (1968a) Pluralit6 des "photordcepteurs" darts l'6piphyse
de l_xtcerta. C R Acad Sci (Paris), S6rie D 267:1047-1050
Collin JP (1968b) L'6pith61ium sensoriel de l'organe pin6al de la
larve gg6e et de l'adulte de Lampetra p[aneri. C R Acad Sci
(PreSs), Sdrie D 267:1768- 1771
Collin IP (1969) Contribution ~t l'6tude de l'organe pin6al. De
l'dpiphyse sensorielle h la glande pin6ale: modalit6s de transformation et implications fonctionneltes. Ann Stat Biol de
Besse-en-Chandesse [Supp] 11:1-359
Collin JP (1971) Differentiation and regression of tile cells of the
sensory line in the epiphysis cerebri. In: Wolstenhohne GEW,
Knight J (cds) The Pineal Gland. Ciba Foundation Symposium. Churchill Livingstonc, Endinburgh London New York,
pp 79-t25
Collin JP, Oksche A (1981) Structural and functional relationships
in the nonmammalian pineal gland. In: Reitcr RJ (ed) The Pineal Gland, vol i: Anatomy and Biochemistry. CRC Press,
Boca Raton, pp 26 67
Korf HW (1994) The pineal organ as a component of the biolegi
cal clock, In: Ann NY Acad Sci 719:13.42
Korf HW, Oksche A, Ekstr6m R Veen T van, Zigler JS, G e ~ L
Stein R Klein DC (1986) S-mltigen immunocytochemistry. In:
O'Brien R Klein DC (eds) Pineal and Retinal Relationships.
Academic Press, New 5%rk, pp 343-355

Oksche A (1965) Survey- of the development and comparative

morphology of the pineal organ. In: AriEns Kappers J, Schad6
JP (eds) Progress in Brain Research, vol 10. Elseviel Amsterdam, pp 3-29
Oksche A (1968) Zur Frage exlraretinaler Ph~Jtorezeptoren im Pinealorgan der VOgel. Arch Anat (Strasbourg) 51:497-507
Oksche A (1971) Sensory anti glandular elements of file pineal organ. In: Wolstenholme GEW, Knight J (eds) The Pineal
Gland. Ciba Foundation Symposium. Churchill Livingstone,
Edinburgh London New York, pp 127 146
Oksche A, Kirschstein H, Kobayashi H, Farmer DS (1972) Electron microscopic and experimental studies of the pineal organ
in the white-crowned spal-row, Zonotrichia leucoph~'s gambelii. Z Zellforsch 124:247-274
Oksche A, Kirschstein H (1969) Elektronenmikroskopische Untersuehungen am Pinea]organ yon Passer domesticus. Z
Zellforsch 102:214-241
Oksche A, Morita Y, Vaupel-von Harnack M (1969) Zur Fcinstrukmr und Funktion dos Pmealorgans der Taube (Columba
livia). Z Zcllforseh 102:1-30
Tamotsu S, Korf HW, Morita Y, Oksche A (1990) Inmaunocytochemical localization of serotonin and photoreceptor-specific
proteins (rod-opsin, S-antigen) in the pineal complex of the
river lamprey, Lampetra japonica, with special reference to
photoneuroendocrine cells. Cell Tissue Res 262:205 216
Zimmennan BL and Tso M (1975) Morphological evidence of
photoreceptor differentiation of pinealocytes in the neonatal
rat. J Cell Biol 66:60-75