the morpho‐anatomy and histology of the pineal complex in a major indian carp, catla catla :...
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The Morpho-anatomy and Histology of the PinealComplex in a Major Indian Carp, Catla catla:Identification of the Pineal Photoreceptor Cells andTheir Responsiveness to Constant Light and ConstantDarkness During Different Phases of the AnnualReproductive CycleR. Dey a; S. Bhattacharya b; S. K. Maitra a; T. K. Banerji ca Department of Zoology, Visva Bharati University. Santiniketan. Indiab Department of Zoology, University of Burdwan. Burdwan. Indiac Department of Anatomy and Neurosciences, University of Texas Medical Branch.Galveston, Texas. USA
Online Publication Date: 12 January 2003To cite this Article: Dey, R., Bhattacharya, S., Maitra, S. K. and Banerji, T. K. , (2003) 'The Morpho-anatomy andHistology of the Pineal Complex in a Major Indian Carp, Catla catla: Identification of the Pineal Photoreceptor Cells andTheir Responsiveness to Constant Light and Constant Darkness During Different Phases of the Annual ReproductiveCycle', Endocrine Research, 29:4, 429 - 443To link to this article: DOI: 10.1081/ERC-120026949URL: http://dx.doi.org/10.1081/ERC-120026949
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The Morpho-anatomy and Histology of the PinealComplex in a Major Indian Carp, Catla catla:
Identification of the Pineal Photoreceptor Cells andTheir Responsiveness to Constant Light and
Constant Darkness During Different Phases ofthe Annual Reproductive Cycle
R. Dey,1 S. Bhattacharya,2 S. K. Maitra,1,# and T. K. Banerji3,*
1Department of Zoology, Visva Bharati University, Santiniketan, India2Department of Zoology, University of Burdwan, Burdwan, India
3Department of Anatomy and Neurosciences, University of Texas Medical Branch,
Galveston, Texas, USA
ABSTRACT
In contrast to mammals in which the pineal gland is a discrete structure situated
dorsally in the brain, the ‘‘pineal gland’’ in teleost fishes is composed of a number
of separate but connected constituent parts, collectively described as the ‘‘pineal
complex.’’ In this paper, we have described the pineal complex in a common Indian
carp, Catla catla, which exhibits an annual reproductive cycle. Attempts have been
made to (a) provide an in-depth description of the structure of the pineal complex; and
(b) identify the photoreceptor cells of the pineal, by exposing the animals to constant
light (LL) and constant darkness (DD). Furthermore, we examined any possible
influence of the reproductive status of the fish on the responsiveness of the pineal
photoreceptor cells in C. catla following exposure to LL and DD. To this end, a total of
#Request for reprints: Dr. S. K. Maitra, Department of Zoology, Visva Bharati University,
Santiniketan 731235, India; E-mail: [email protected].*Correspondence: Dr. T. K. Banerji, Department of Anatomy and Neurosciences, University of Texas
Medical Branch, 301 University Blvd., Galveston, TX 77555-1069, USA; E-mail: [email protected].
ENDOCRINE RESEARCH
Vol. 29, No. 4, pp. 429–443, 2003
DOI: 10.1081=ERC-120026949 0743-5800 (Print); 1532-4206 (Online)
Copyright # 2003 by Marcel Dekker, Inc. www.dekker.com
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four experiments were carried out during the four different phases of the annual
reproductive cycle that is characteristic of this species. Each of these four experiments
was carried out for a period of 30 days after which the fishes were sacrificed, different
parts of the pineal complex were dissected out, and processed for histological and
karyometric studies. Our results showed that the pineal complex in this species is
composed of three separate but connected parts, (a) an end vesicle (EV); (b) a dorsal
sac (DS); and (c) a long and thin pineal stalk (PS) that attaches the EV to the DS.
Detailed karyometric and histo-morphologic studies following exposure of the animals
to DD and LL showed that constant darkness led to a stimulatory effect on the pineal
photoreceptor cells of the EV as evident from a significant increase in the nuclear
diameter. In contrast, the nuclear diameter of the photoreceptor cells in animals
subjected to constant light showed a significant reduction. Furthermore, the observed
cellular changes in the EV of fish exposed either to LL or DD were independent of the
stage of the gonadal cycle. The apparent lack of any cellular responses either in the PS,
or in the DS, following exposure to LL and DD, suggests that in C. catla the
photoreceptor cells are located only within the epithelial lining of the EV and that
these cells respond in a manner similar to mammalian pinealocytes when subjected to
comparable photoperiod-induced experimental conditions.
Key Words: Pineal complex; Teleosta; Photoreceptor cells; Constant light and
darkness.
INTRODUCTION
The role of the pineal gland as a neuroendocrine transducer in mammals is now well
established. It exhibits circadian rhythmic activities, responds to photoperiodic stimuli, and
transmits these stimuli to modulate the functional status of many other endocrine glands
(1–4). The pineal gland is also known to have an anti-gonadal effect in mammals.
The existence of pineal’s anti-gonadal effects in some sub-mammalian species, especially
those characterized by a defined annual reproductive cycle, has also been reported (5–7).
While the pineal is a discrete endocrine gland present dorsally in the epithalamic area of
the brain in mammals, in lower vertebrates the pineal organ has been reported to show a
wide range of structural variabilities (8,9). From an evolutionary perspective, it becomes
readily apparent that the structure and function of the pineal gland have undergone a clear
transformation from being primarily a sensory organ in lower vertebrates to an important
endocrine gland in mammals. It is noteworthy that the pineal organ in some lower
vertebrates exhibits both sensory as well as secretory functions (10–12). Evidence has also
been presented that the pineal organ in some lower vertebrates is directly photosensitive
(13). The teleostean pineal organ has been suggested to serve as an important component
of the photo-neuroendocrine system transmitting and transducing photic signals into
neuroendocrine messages (14–17).
In teleosts, the pineal gland is usually described as a pineal complex consisting of
several components distributed both within and outside the brain (17,18). The piscine
pineal complex has been reported to be composed of three separate parts that are
connected to each other—an end vesicle (EV), a pineal stalk (PS) of varying length,
and the saccus dorsalis, the dorsal sac (DS). Recent studies (19,20), utilizing a number of
teleost species, demonstrated that there is striking variability with regard to the fine
structure and histo-morphology of the pineal complex. A perusal of the present literature,
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however, reveals that very little information is available on the pineal organ in teleost fishes
from the Indian sub-continent. Therefore, in a recent study (21) we utilized the major
Indian carp, C. catla, a species with great commercial interest, and investigated the basic
structure of the pineal complex. In that study we also examined whether or not, as in
mammals, the pineal organ of this species has an anti-gonadal effect.
In the present study, utilizing the same species of carp, C. catla, we report an in-depth
karyometric measurements of the various parts of the pineal complex, with special
attention being directed in identifying the photoreceptor cells of the pineal organ. To
this end, we have utilized a number of experimental approaches such as exposure of the
animals to constant light and constant darkness—conditions known to modulate and
influence the functional status of pinealocytes in many vertebrates (1,22–24). Furthermore,
given the well-known variations in the responsiveness of the pineal organ in vertebrates to
the reproductive status of the animals (7,25), the responses of the pineal photoreceptor
cells following exposure to photoperiodic alterations were examined throughout the annual
reproductive cycle of this species.
MATERIALS AND METHODS
The campus of the University of Burdwan (Lat. 23�140N, Long. 87�510E), West
Bengal, India, where the experiments were carried out, is surrounded by numerous large
lakes. Sexually adult carp, C. catla, weighing between 600 and 700 g were captured by
local professional fishermen utilizing dragnets, and were promptly transported to the
laboratory. Since there is no sexual dimorphism in this species, and thus males and females
are not distinguishable externally, fish of both sexes were used initially. However, at the
termination of each experiment, only females were considered for our investigation.
Following arrival in the laboratory, the fish were immediately transferred to aquaria
[2.00 m (L)� 80.00 cm (W)� 70.00 cm (H)] equipped with continuous water flow and
aeration system. Subsequently, following a period of acclimation, they were divided into
three experimental groups and were exposed to three different photoperiodic conditions:
Group NP (control; normal photoperiod, LD: 12:12h; normal daily day–night cycle with
12 hours of light and 12 hours of darkness); Group LL (continuous light, LD: 24:00h); and
Group DD (continuous darkness, LD: 00:24h). For the fish in the LL group, continuous
light was provided with 40 watt white fluorescent lamps placed horizontally at about 50 cm
above the aquarium water surface. The intensity of the light at the upper water surface was
about 150 lux. Aquaria that contained the fish for continuous darkness were maintained in
a somewhat distant location within the laboratory, and were covered by protective light-
proof devices.
Catla catla of both sexes characteristically exhibit one breeding cycle per year. In
a recent communication (21) we have reported four specific phases of the annual
reproductive cycle in the males. These phases are: (1) preparatory (January–April);
(2) pre-spawning (May–June); (3) spawning (July); and (4) post-spawning (August–
December). Our ongoing studies (unpublished data) also reveal that, just like the males,
in female C. catla there is also an annual reproductive cycle, and that there are four
reproductive phases that coincide almost exactly with those of the males.
There were three objectives for our present study. First, to investigate the detailed
morpho-anatomy and histology of the different components of the pineal complex in
Pineal Photoreceptor Cells in Catla catla 431
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C. catla. Second, to identify the pineal photoreceptor cells of this species by exposing
them to constant light and constant darkness. Third, to examine whether or not the
responses of the photoreceptor cells in the pineal organ, following alterations in the
photoperiodic regimen, differ depending on the phase of the reproductive cycle. To this
end, three experimental groups were utilized (NP, LL, and DD), and a total of four
experiments were conducted representing the four phases of the reproductive cycle
mentioned above. Each of these four experiments lasted for 30 days. As indicated earlier,
some of the phases of the reproductive cycle in the females normally extend over more
than a month; therefore, the 30-day-long experiments were conducted during the
following months representing each of the phases of the gonadal cycle: preparatory
(January–March); pre-spawning (April–May); spawning (July); and post-spawning
(August–December). At the completion of each experiment, animals were sacrificed by
quick decapitation. The fish exposed to continuous darkness were sacrificed under dim red
light, which does not appear to affect pineal function (18). Following sacrifice of the
animals, the skull was dissected open and the entire pineal complex from each animal was
collected in two parts, (a) the EV with a part of the PS, and (b) the DS and the part of the
brain where the DS is attached. The tissues were fixed in Bouin’s solutions, and following
routine histological procedures 4 mm sections were obtained. Sections were routinely
stained in Masson’s trichrome.
Measurement of nuclear diameter has been shown to be a reliable index for the
evaluation of cellular responses of the pineal to diverse experimental situations, including
altered photoperiodic conditions (26,27). Therefore, in the present experiment, the nuclear
diameter of the pineal parenchymal cells were measured and compared among the various
experimental groups. To this end, at least 100 nuclei of each of the light and dark cells
were randomly selected from five sagital sections of the EV. Similarly, about 100 nuclei
were measured from the epithelial lining of PS and DS. In either case, the nuclear
diameters were measured in a bright-field PRIOR (UK) microscope under oil immersion
using 15 ocular� 100 objective lenses along with an ocular micrometer scale. The values
obtained were then converted to millimeter with the help of a calibrated ocular micrometer.
In a recent communication (21) we have provided a preliminary report on the structure of
the pineal organ in C. catla. We now present an in-depth investigation of the pineal organ
of this species, special attention being directed to the karyometric measurement of the
nuclear diameter of the epithelial cells in different parts of the pineal complex, and
especially those in the EV parenchyma where photoreceptor cells are located.
For statistical analysis of our data, one-way analysis of variance (ANOVA) was
followed by Student’s t-test.
RESULTS
Morpho-anatomy of the Pineal Complex
The pineal complex in C. catla is composed of three component parts, (a) an antero-
laterally elongated, dorso-ventrally flattened vesicular part, the EV; (b) a long, thin, and
hollow PS; and (c) a DS, which is highly folded and located on the dorsal surface of
the brain (Fig. 1). We conducted detailed karyometric measurements of each of these three
parts of the pineal complex following various experimental manipulations. However, since
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photoreceptive cells were found only in the epithelial lining of the EV, karyometric data on
nuclear diameter changes of EV epithelial cells following various experimental manipula-
tions are included in Table 1.
The End Vesicle
The EV is the anterior-most part of the pineal complex (Fig. 1) and is lodged in a
shallow depression or concavity in the ventral part of the cranial roof. It lies far rostral to
the brain at the level of the paired lateral eyes and is lodged underneath the highly
specialized region of the skull that is slightly translucent and where ossification and
pigmentation are comparatively scant. The mean values for the various dimensions of the
EV were: 1.60� 0.24 mm in antero-posterior length; 7.78� 1.25 mm in transverse length;
and 311.10� 62.46 mm in dorso-ventral thickness. The EV parenchyma is highly folded
and the infoldings are present in both the dorsal and ventral layers. The underlying stroma
of the lamina propria is highly vascularized (Fig. 2). We found two types of cells in the EV
parenchyma; (a) cells that stain rather lightly and characterized by the presence of
Figure 1. A diagram (not to scale) illustrating the three constituent parts of the pineal complex in
C. catla and their relationship to the skull and various parts of the brain. Key: EV, end vesicle; PS,
pineal stalk; DS, dorsal sac; C, cartilage; VT, velum transversum; HC, habenular commissure; SCO,
subcommissural organ; NH, habenular nucleus. [The diagram is an adaptation of a figure presented
in our earlier communication (see Ref. 21)].
Pineal Photoreceptor Cells in Catla catla 433
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Ta
ble
1.
Kar
yo
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ric
mea
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er(m
m)
of
EV
epit
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ial
cell
sin
C.
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afo
llow
ing
exp
osu
reto
nat
ura
lp
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top
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d(N
P),
LL
,an
dD
Dd
uri
ng
the
dif
fere
nt
ph
ases
of
the
ann
ual
rep
rod
uct
ive
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e.
Gro
up
Ph
ase
of
rep
rod
uct
ive
cycl
e
NP
aL
La
DD
a
Ph
oto
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pto
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llS
up
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tore
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Su
pp
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cell
Ph
oto
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pto
rce
llS
up
po
rtin
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ll
Pre
par
ato
ry
(Jan
uar
y–
Mar
ch)
4.7
3�
0.0
23
.34�
0.0
64
.49�
0.0
5b
3.3
6�
0.0
55
.59�
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3.3
2�
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6
Pre
-sp
awn
ing
(Ap
ril–
May
)
4.5
8�
0.0
53
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24
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2c
3.2
2�
0.0
94
.93�
0.0
8c
3.2
2�
0.1
1
Sp
awn
ing
(Ju
ly)
4.4
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0.0
53
.34�
0.9
4.2
3�
0.0
7d
3.2
2�
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04
.69�
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3d
3.2
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2
Po
st-s
paw
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(Au
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Dec
emb
er)
4.9
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73
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9�
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6
aT
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ied
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and
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prominent apical cytoplasmic projections that reach into the lumen; and (b) smaller cells
that are comparatively darkly stained that have no luminal contact. Both of these cell types
are in clusters and there is a substantial intermingling between them (Fig. 3).
Suggestions have been made by some authors that these two cell types represent the
pineal photoreceptor and supporting cells respectively (20,28–30).
The Pineal Stalk
The PS is a long and thin tubular structure with a clearly discernible lumen, which is
continuous with the third ventricle (Fig. 1). The PS arises from the diencephalic roof
between the habenular nuclei and posterior commissure, runs anterodorsally passing
through the adipose connective tissue to the EV. Histologically, the cellular elements of
the PS exhibit some differences in that there are no infoldings of the epithelial lining in the
PS (Fig. 1). The mean total length of the PS was 11.20þ 1.02 mm, and its mean diameter
measured 237.50þ 35.77 mm.
The Dorsal Sac
The roof of the diencephalon forms an evagination in between the habenular
commissure and velum transversum and is called the DS (Fig. 1). It is a hollow saccular
Figure 2. Low power microphotograph of a section through the EV of the pineal complex in
C. catla showing the organization of the epithelial lining on the dorsal and ventral aspects. Note the
large central lumen, the infoldings of the parenchyma and the vascular beds in the underlying
connective tissue of the lamina propria. The tissue was obtained from control animals exposed to
normal photoperiod and sections were stained in Masson’s trichrome. 200�.
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structure and is thrown into several large folds, which surround the base of the PS up to
one-third of its length. A prominent blood vessel (vena cerebri magna) runs parallel to the
PS and forms a plexus surrounding the dorsal and ventral surfaces of the EV. The cells of
the DS appear to be long, ciliated, and columnar. The lumen of the DS has open
communication with the third ventricle (Fig. 1). The mean dorso-ventral height of the
DS was 1636.26� 28.53 mm, and the measurement of its width gave a mean value of
786.16� 15.40 mm.
Figure 3. High power microphotograph of a Masson’s trichrome stained section through the EV of
the pineal complex in the control C. catla exposed to normal photoperiod. Note a portion of the
folded epithelial lining on the dorsal and ventral aspects of the EV with part of its enclosed central
lumen. The photoreceptor cells with prominent cytoplasmic projections that extend into the lumen
are clearly seen. Many of these cytoplasmic processes are long and appear to be clumped. 800�.
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Responsiveness of the Pineal Complex Under Altered
Photoperiodic Conditions
As indicated earlier, our primary goal was to examine whether or not altered
photoperiod such as constant light or constant darkness had any effect on the EV
epithelial cells. We also wanted to examine whether or not any altered photoperiod-
induced changes were secondarily influenced by the phase of the annual reproductive
cycle. To achieve these goals, a total of four experiments were carried out, each
representing a specific phase of the reproductive cycle of this species. Each of the four
experiments lasted for 30 days, and included all three experimental groups (NP, LL, and
DD). Our results showed that exposure to both continuous light and darkness led to
significant cellular responses. Interestingly, these changes were evident only in the
epithelial cells of the EV; no significant changes were observed in the cells of either
the PS or the DS. Karyometeric measurements of the nuclear diameter of the EV cells
following exposure to photoperiodic alterations are incorporated in Table 1. Other
important observations are as follows.
Group NP: Control (LD: 12:12h)
Preparatory phase (January–March): During this period, the EV parenchyma exhibited
infoldings and the photoreceptor cells showed prominent cytoplasmic extensions
deep into the lumen.
Pre-spawning phase (April–May): The epithelial cells of the EV parenchyma appeared
thin, and the cytoplasmic extensions from these cells were reduced compared to
those found in the previous phase.
Spawning phase (July): Notably, during this phase the EV parenchymal thickness in
both the dorsal and ventral aspects became significantly reduced and appeared
practically flat. The apical cytoplasmic processes were practically absent.
Post-spawning phase (August–December): During this period the EV parenchymal
thickness increased and the epithelial cells lining the lumen showed prominent
cytoplasmic extensions deep into the lumen.
Group DD: Constant Darkness
The nuclear diameters of the photoreceptor cells in the EV parenchyma were
significantly increased when compared with those of the respective control (NP) groups
(Table 1). Furthermore, the epithelial cells underwent a marked proliferation resulting in a
significant increase in the overall thickness of the epithelium, it being more prominent in
the ventral surface of the EV. Cytoplasmic extensions of these cells were long and, quite
often they appeared as clumps within the lumen of the EV (Fig. 4). The responses of
these epithelial cells in EV to constant darkness were found to be identical irrespective of
the phase of the annual reproductive cycle.
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Group LL: Constant Light
The responses of the EV epithelial cells exposed to constant light were just the reverse
of the changes noted following constant darkness. As a whole, the EV epithelium appeared
very flat, the mean nuclear diameter of these cells was significantly reduced (Table 1), and
the epithelial cells were practically devoid of any cytoplasmic projections (Fig. 5).
Also, as noted in the animals of the DD group, the above cellular changes due to
constant light were evident irrespective of the phase of the annual reproductive cycle of
this species.
DISCUSSION
While the present study on the anatomic location and structure of the pineal complex
in C. catla confirms the findings presented in our recent publication (21), it also extends
further in providing a detailed account of the pineal complex, especially with regard to
karyometric measurements of the three components of the pineal complex—the EV, PS,
and DS. Our data show that the overall organization of the pineal complex of this species
has a close similarity with that of few other cyprinid fishes (31,32). In C. catla, the pineal
EV lies far rostral to the brain at the level of the paired lateral eyes and is lodged in a
Figure 4. High power microphotograph of a Masson’s trichrome stained section through the EV of
the pineal complex in C. catla exposed to constant darkness. A portion of the ventral aspect of the
EV is shown here with part of the parenchymal folding. The lumen is seen at the upper part of the
photograph. Exposure to constant darkness had a stimulatory effect on the pineal photoreceptor cells
whose nuclear diameters showed a significant increase (Table 1). The long apical cytoplasmic
projections of the photoreceptor cells appear clumped in the lumen. 800�.
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concavity of the cranial roof. This is an area that is less pigmented and more translucent,
and thus conducive for penetration to light and its passage to the photoreceptor cells
located within the epithelial lining of the EV. That light does penetrate into the brain of
mammals has been clearly shown (33).
A number of earlier studies have suggested that the cells distributed in the epithelial
lining of the EV represent the photosensory elements of the fish pineal, the structural and
Figure 5. High power microphotograph of a Masson’s trichrome stained section of the EV of
C. catla exposed to LL. A portion of the ventral and dorsal aspects of the EV is seen here with the
large central lumen. Note the marked thinning of the EV epithelial lining and a reduction in the size
of the epithelial photoreceptor cell whose nuclear diameters showed a significant reduction (Table 1).
The apical cytoplasmic projections of the photoreceptor cells also appear to be practically non-
existent. 800�.
Pineal Photoreceptor Cells in Catla catla 439
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functional homologue of the mammalian pinealocytes (13,14,16,34). The arrangement of
the pineal parenchyma shows wide variations among teleosts and on this basis three main
types have been described (18): (a) wide-spaced; (b) intermediate; and (c) compact types.
In our present experimental subject, the notable infoldings of the EV parenchyma, its
typical cellular arrangements, the presence of extensive vascular beds in the lamina propria
below the epithelium, and other features seemingly place the pineal of the present species
in the category of the ‘‘wide-spaced type.’’ In an attempt to identify the photoreceptor cells
of the pineal complex in fish, both light and electron microscopic studies have been
utilized by a number authors (8,15,19,20,31). Many of these studies suggest that the pineal
photoreceptor cells are represented by those cells in the EV parenchyma that mostly line
the lumen of the EV, and are provided with long cytoplasmic processes that project deeply
into the lumen. In our recent communication (21) in which we used male C. catla, we too
observed these cells and our data obtained from the responses of these cells during the
various reproductive phases of the annual cycles strongly suggest that these epithelial cells
do indeed respond like pineal photoreceptor cells.
One of the major objectives of the present study was to provide more direct
experimental evidence in identifying the photoreceptor cells in C. catla. To this end, we
exposed the experimental subjects to continuous light and continuous darkness, conditions
under which mammalian pinealocytes are known to respond (1). Our experiments on the
effects of altered photoperiodic regimens such as constant light and constant darkness
provided important insights in identifying the photoreceptor cells in the pineal complex of
C. catla. It is noteworthy that the observed cellular changes due to photoperiodic
manipulations were evident only in the parenchymal cells of the EV; no changes were
observed in cells of either the PS or the DS. These are the cells that characteristically
possess long cytoplasmic extensions that normally project deep into the lumen of the EV.
Apparently, other authors have also observed cells with similar features, and have
suggested that these cells represent the photoreceptor cells of the piscine pineal organ
(10,16,21,23,35).
A number of authors have shown that measurement of nuclear diameter can be used as
a reliable index for the evaluation of cellular responses of the pineal when exposed to
altered photoperiodic conditions (5–7,26,27,36,37). Clearly, in the present experiment, the
nuclear diameter of the photoreceptor cells of the EV epithelium showed marked
alterations following exposure to LL or DD. Exposure to DD was stimulatory for these
cells, resulting in significantly increased nuclear diameters and highly prominent apical
cytoplasmic processes, while LL rendered the epithelial lining flat with significantly
reduced nuclear diameter (Table 1) of these cells, and near-atrophied apical cytoplasmic
processes (Fig. 5).
Our present experiments, therefore, provide direct experimental evidence in identify-
ing the photoreceptor cells in this species. It is also important to note that the responses of
the photoreceptor cells, evident from characteristic changes in the nuclear diameter
following exposure to LL and DD, did not significantly differ during the different phases
of the reproductive cycle of this species. In other words, photoperiodic cues were the
overwhelming determinant factors in producing the cellular responses in the EV photo-
receptor cells.
In conclusion, our experiments confirm the findings reported in our recent commu-
nication (21), and have extended further our understanding on the structure and function of
the pineal complex of C. catla, especially with regard to its anatomic location and
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karyometric histo-morphology. Furthermore, our data provide important experimental
evidence in identifying the pineal photoreceptor cells, the presumptive homologue of the
pinealocytes in mammals.
ACKNOWLEDGMENT
We gratefully acknowledge the financial assistance from the Indian Council of
Agricultural Research, Government of India, New Delhi, through a research grant
(F.No. 4-(40)=96-ASR-I) to SKM.
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