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Pinopsin and photoreception in the pineal organ of the domestic turkey during post-hatching development
Micron 126 (2019) 102749
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Pinopsin and photoreception in the pineal organ of the domestic turkey during post-hatching development
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Marcela Petrusewicz-Kosińska, Barbara Przybylska-Gornowicz , Magdalena Prusik, Natalia Ziółkowska, Bogdan Lewczuk Department of Histology and Embryology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego 13, 10-719 Olsztyn, Poland
A R T I C LE I N FO
A B S T R A C T
Keywords: Pineal organ Extraocular photoreception Pinopsin Melatonin Development Birds
The avian pineal organ is photosensitive because of the presence of photopigments, of which pinopsin seems to be one of the most important. This organ is subject to far-reaching changes during post-hatching development, but evidence regarding pinopsin presence and direct photoreception during this time is lacking. This study was carried out to demonstrate the following: 1) the structures showing immunoreactivity to pinopsin in the turkey pineal organ, 2) the changes of these structures during development, 3) the pinopsin localization in pinealocytes in monolayer cultures, and 4) the role of direct photoreception in the regulation of melatonin secretion in pineal organs in adult turkeys. Pinopsin immunoreactivity was localized in the apical extensions of columnar cells limiting the follicular lumen, in fiber-like structures located between columnar cells in the inner part of follicle wall, in string-shapes or small spherical structures distributed in the outer part of follicle wall and in amorphous material inside the follicle lumen. In young birds, immunoreactivity was also sporadically noted in cell bodies of rudimentary receptor pinealocytes. The distribution of pinopsin showed prominent age-dependent changes, including a subsequent increase in pinopsin-positive structures in the outer part of the follicle wall and a prominent reduction in the number and size of positive apical extensions in 40- and 56-week-old turkeys. These data demonstrate that the role of secretory pinealocytes in pineal photoreception increases with age. In monolayer cultures, all pinealocytes showed strong reactions in club- or bulbous-shaped prolongations. The pineal organs of adult birds were less sensitive to light exposition at night than those of young turkeys, which points to differences in light sensitivity between rudimentary receptor and secretory pinealocytes. However, direct photoreception could play an important role in the regulation of melatonin secretion in adult turkeys.
1. Introduction Avian pinealocytes possess photoreceptive capability, endogenous circadian oscillator and melatonin synthesizing machinery (Nakahara et al., 1997). The predominant pineal photoreceptive molecule in birds, pinopsin, controls the daily melatonin output (Takanaka et al., 1998; Holthues et al., 2005; Mano and Fukada, 2007). Pinopsin belongs to the nonvisual opsins, which are detected in extraocular photoreceptive sites localized in the pineal complex and other structures of the brain in nonmammalian vertebrates (Bertolucci and Foa, 2004). Pinopsin was initially cloned from the chicken pineal organ (Okano et al., 1994), and its expression is probably limited to birds. Pinopsin possesses a rhodopsin-like heptahelical primary structure with a chromophore of 11-cis-retinal (Okano and Fukada, 1997) and exhibits sensitivity to blue light, with maximum absorbance at
approximately 470 nm (Okano et al., 1994; Nakamura et al., 1999). It is a member of the GTP-binding protein-coupled receptor family, and the light signal captured by pinopsin is most likely transduced by heterotrimeric G-protein. The colocalization of G-protein α-subunits, the rod transducin Gt1α and Gq/11α, with pinopsin suggests that these subunits are functionally coupled with this opsin (Matsushita et al., 2000). Although the precise role of pinopsin has not been finally elucidated, there is evidence of its mediation of the acute effect of light on melatonin secretion by the Gt1α pathway (Kasahara et al., 2000). Moreover, colocalization of Gq/11α with pinopsin suggests that this photopigment is also involved in entrainment of the intrinsic circadian oscillator (Matsushita et al., 2000; Mano and Fukada, 2007). To date, the presence and localization of pinopsin in the pineal organ of birds have been studied only fragmentarily in a few species, such as young chicken (Hirunagi et al., 1997; Okano et al., 1997), adult
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Corresponding author. E-mail addresses: [email protected] (M. Petrusewicz-Kosińska), [email protected] (B. Przybylska-Gornowicz), [email protected] (M. Prusik), [email protected] (N. Ziółkowska), [email protected] (B. Lewczuk). https://doi.org/10.1016/j.micron.2019.102749 Received 2 August 2019; Received in revised form 2 September 2019; Accepted 2 September 2019 Available online 03 September 2019 0968-4328/ © 2019 Elsevier Ltd. All rights reserved.
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were anesthetized with isoflurane and killed by decapitation between 08:00 and 09:00 at the age of 2 days, 2, 10, 20, 30, 40 and 56 weeks. The pineal organs with adjacent parts of the brain were immediately removed and prepared for immunohistochemical study. Additionally, 10 pineal organs of 20-week-old turkeys were collected for the preparation of a monolayer cell culture, and 30 pineal organs were obtained from birds at the ages of 10 weeks (n = 12) and 40 weeks (n = 18) for a superfusion culture. All experimental procedures involving animals were carried out in compliance with Polish legal regulations determining the terms and methods of performing experiments on animals.
pigeons (Okano et al., 1997) and quail embryos (Yamao et al., 1999). Pinopsin immunoreactivity has been found in the pear-shaped apical protrusions of follicular cells extending into the follicle lumen (Hirunagi et al., 1997; Okano et al., 1997; Yamao et al., 1999) and in long slender structures in the parafollicular pineal parenchyma (Hirunagi et al., 1997). The avian pineal organs vary considerably in their anatomical forms (Quay and Renzoni, 1967; Vollrath, 1981) and histological organization (Vollrath, 1981; Ohshima and Matsuo, 1984; Sato, 2001). The structure of this organ also undergoes prominent changes during ontogenesis (Sato et al., 1990; Ohshima and Hiramatsu, 1993; Sato, 2001; Przybylska-Gornowicz et al., 2005; Petrusewicz-Kosińska et al., 2019). It should be stressed that comparative studies on the presence and distribution of pinopsin are still lacking, and as a consequence, very little is known about the localization of this photomolecule in different forms of the avian pineal organ. Literally nothing is known about changes in the distribution of this photomolecule during the posthatching transformation of the pineal organ. In our previous studies, we have traced morphological aspects of developmental processes of the domestic turkey pineal organ during the period from one day until one year of post-hatching life (PrzybylskaGornowicz et al., 2005; Petrusewicz-Kosińska et al., 2019). The turkey pineal organ retains a follicular structure throughout the entire investigated period and comprises two types of pinealocytes, rudimentary photoreceptor pinealocytes and secretory pinealocytes. Rudimentary photoreceptor pinealocytes compose a part of the follicle wall surrounding the lumen and develop until the age of 20 weeks. This process leads to the appearance of extremely well-developed cells with prominent apical prolongation and stratified organelle distribution in turkeys at the ages of 20 and 30 weeks. The stratified distribution of organelles is similar to the compartmentalization of retinal photoreceptors and pineal photoreceptors of lower vertebrates, which are involved in phototransduction (Collin et al., 1986). Then, rudimentary photoreceptor cells show pronounced regressive changes in 40- and 56-weekold birds; the number and size of the organelles are distinctly reduced, and apical protrusions are reduced. In contrast to rudimentary photoreceptor pinealocytes, secretory pinealocytes lack contact with the follicle lumen and polarization. Their number increases during the postnatal development, and they form a prominent outer part of the follicle wall in 40- and 56-week-old turkeys (Petrusewicz-Kosińska et al., 2019). The changes in the organization of the follicles suggest the reduction of photoreceptor capability of the turkey pineal after sexual maturation. The results of morphological studies prompted us to ask the question of whether the observed changes also apply to pinopsin, the main sensory input system of the avian pineal organ. In this study, we used immunochemical staining to examine the presence and distribution of pinopsin in the pineal organs of turkeys at various stages of posthatching life. We also prepared a monolayer culture of turkey pineal cells to check whether the localization and morphology of pinopsinpositive structures in pinealocytes is determined by their contact with the follicle lumen or if it is genetically programmed by the type of cell (rudimentary receptor or secretory pinealocyte). Finally, we compared in vitro the effect of light exposure during scotophase on melatonin secretion from the pineal organs of 10-week-old and 40-week-old turkeys as well as their ability to entrain secretory activity to a reversed light-dark cycle.
2.2. Chemicals The mouse monoclonal antibody against pinopsin was obtained from Prof. Y. Fukada and Prof. T. Okano from the University of Tokyo, Japan. A procedure for preparation of the antibody as well as validation of its specificity was described in detail by Okano et al. (1997). The rabbit primary antiserum against chicken N-acetylserotonin O-methyltransferase (ASMT) was a gift of Dr Pierre Voisin, University of Poitiers, France. A procedure for preparation and characterization of the antibody as well as validation of its specificity was described in detail by Voisin et al. (1988). Secondary antibodies coupled with AlexaFluor 488 or AlexaFluor 594 and DAPI were purchased from Molecular Probes (Eugene, OR, USA). The anti-melatonin antibody Prospect 6C was kindly provided by Dr Andrew Foldes (Division of Animal Production, CSIRO, Blacktown, Australia). 3H-melatonin was purchased from PerkinElmer (Waltham, MA, USA). Gelatin was obtained from Merck (Billerica, MA, USA) and paraformaldehyde from Polysciences, Inc. (Warrington, PA, USA). Medium 199 with Earle’s salt and HEPES, Hank’s balanced sodium salt, Earle’s balanced sodium salt, Dulbecco's modified eagle medium, antibiotic-antimycotic solution, fetal bovine serum, collagenase IV, DNA-se, poly-L-lysine and all other chemicals were purchased from Sigma (St. Louis, MO, USA). 2.3. Immunohistochemical studies The pineal organs were fixed in a 4% paraformaldehyde solution in 0.1 M phosphate buffer (pH = 7.4) for 45 min. Then, the tissues were rinsed with phosphate buffer, cryoprotected in 25% sucrose solution, frozen and cut into 10 μm thick sections using a Microm HM 560 cryostat (Microm, Walldorf, Germany). The sections mounted on glass slides were subjected to an immunohistochemical procedure using the mouse monoclonal antibody against pinopsin (working dilution of 5 μg/ ml). The primary antibodies bound to the antigens present in the tissue slides were visualized by means of secondary goat anti-mouse IgG antiserum coupled with AlexaFluor 488. After intense washing, cell nuclei were stained in DAPI solution, and the sections were sealed in a mixture of polyvinyl alcohol and glycerine with 2% DABCO. The sections were examined using a fluorescence microscope equipped with a digital camera (Carl Zeiss, Oberkochen, Germany). The control procedures were performed with omission of the primary antisera. 2.4. Monolayer cell culture and immunocytochemistry 2.4.1. Cell culture The pineal organs of 20-week-old turkeys were collected in Hank’s Balanced Sodium Salt containing penicillin (1000 Ul/ml), streptomycin (1 mg/ml) and amphotericin B (2.5 μg/ml) and transported to the cell culture laboratory within 10 min after the animals’ death. The organs were cut into several parts and subjected to enzymatic digestion performed at 37 °C for 60 min in a solution containing collagenase IV (2 mg/ml) and DNase (4 μg/ml) in Earle’s balanced sodium salt. After dispersion, the cells were washed three times with culture medium containing 10% bovine fetal serum and then suspended in Dulbecco's Modified Eagle Medium with fetal calf serum (10%), penicillin (100 Ul/
2. Materials and methods 2.1. Animals and pineal glands The study was performed on females of the domestic turkey (Meleagris gallopavo). The birds (apart from those that were newly hatched) were housed under a cycle consisting of 12 h of light and 12 h of dark (light from 07:00 to 19:00). The turkeys (six for each age group) 2
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Fig. 1. Immunohistochemical localization of pinopsin in the pineal organ of two-day-old turkey. A. Pinopsin immunoreactive structures. B. DAPI nuclear staining; C. Differential interference contrast; D – imposition of figures A, B, C. Note presence of positive reaction exclusively in the apical protrusions extending into the follicle lumen (A), the regular arrangement of cell nuclei (B) and the shape of cells (C, D).
ml), streptomycin (0.1 mg/ml) and amphotericin B (0.25 μg/ml). The cells were placed in culture plates covered by poly-L-lysine. Culture was performed at 38 °C under a 12 h light:12 h dark cycle (12 L:12 D) with fluorescent illumination between 09:00 and 21:00. The medium was changed 48 h after the plating and then every 12 h. Cell morphology during the culture was investigated using inverted microscope equipment with phase and relief contrasts (Carl Zeiss, Oberkochen, Germany).
secondary goat anti-mouse IgG antiserum coupled with AlexaFluor 488 and the anti-rabbit IgG coupled with Alexa 594. The control procedures were performed with omission of the primary antisera. The sections were examined using an inverted fluorescence microscope equipped with a digital camera (Carl Zeiss Microscopy, Oberkochen, Germany). 2.5. Superfusion culture and melatonin secretion study 2.5.1. Superfusion culture The pineal organs were collected in Medium 199 containing penicillin (100 Ul/ml), streptomycin (0.1 mg/ml) and amphotericin B (0.25 μg/ml) and transported to the cell culture laboratory within 10 min after the animals’ death. Next, the pineal organs were covered with nylon mesh and placed in culture chambers. The lower pool of each chamber was connected to the vessel with culture medium. The
2.4.2. Immunocytochemistry After 7 days, the cell cultures were fixed with 4% paraformaldehyde for 15 min and subjected to a double immunohistochemical procedure using mouse monoclonal antiserum against pinopsin (5 μg/ml) and rabbit primary antiserum against chicken ASMT (working dilution 1:500). The antigen-antibody complexes were revealed by the 3
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Fig. 2. Immunohistochemical localization of pinopsin in the pineal organ of two-week-old turkey. A. Pinopsin immunoreactive structures. B. Imposition of figure A with DAPI nuclear staining. Note presence of fiber-like structures located between columnar cells in the inner part of the follicle wall.
Fig. 3. Immunohistochemical localization of pinopsin in the pineal organ of ten-week-old turkey. A. Pinopsin immunoreactive structures. B. Imposition of figure A with DAPI nuclear staining and differential interference contrast. Note presence of positive structures in the outer part of the follicle wall (arrows).
2.5.2. Experimental procedures Two experiments were performed. In experiment I, pineal organs obtained from 10-week-old and 40-week-old turkeys were incubated under a 12 L:12 D cycle during the first two days of the experiment and then in continuous darkness for two days. The explants of both age groups were exposed to light (fluorescent lamp, 100 lx) beginning at 01.00 for 30 (n = 3), 60 (n = 3) or 120 (n = 3) minutes during the second day of the experiment. The control pineal organs (n = 3) were not exposed to the light during scotophase. The second experiment was performed on two groups of the pineal organs collected from 40-week-old turkeys. The first group (n = 3) was incubated under a 12 L:12 D cycle and the second group (n = 3) under a reversed cycle of 12 D:12 L during the first three days of the
upper pool of the culture chamber was attached to a multichannel peristaltic pump (Cole Parmer, Vernon Hills, IL, USA) and a manual fraction collector. The chamber was superfused at a rate of 0.1 ml/min with Medium 199 containing penicillin (100 Ul/ml), streptomycin (0.1 mg/ml) and amphotericin B (0.25 μg/ml), which was continuously gassed with a mixture of 95% O2 and 5% CO2. The incubation was performed at 38 °C in a water bath (Julabo, Seelbach, Germany). The chambers were illuminated with a full-spectrum fluorescent lamp providing light with an intensity of 100 lx at their surface. They were covered with light-proof plastic sheets during scotophase and with similar translucent sheets during incubation during photophase. Medium samples were collected every 30 min and frozen at −20 °C until melatonin assays were carried out. 4
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Fig. 4. The pineal organ of a twenty-week-old turkey. A. Pinopsin immunoreactive structures. B. DAPI staining. C. Differential interference contrast, D. Imposition of figures A, B, C. Note strong immunoreactivity situated in amorphous material in the lumen of follicle (star), in the apical prolongations of columnar cells (double arrow) and in string-shaped or small spherical structures in the outer part of follicle wall (arrows). The nuclei created a compact layer in the inner part of the follicle wall and were loosely dispersed in the outer part.
(LSD) was applied as a post hoc procedure. To assess the changes in a course of the melatonin secretion rhythm, the middle of each peak was determined as the point (daily time) halfway between the point at which secretion increased to 75% of the maximum value and the point at which secretion decreased to 75% of the maximum value. To perform statistical analysis, minutes were expressed as tenths of hours (e.g., 15:15 = 15.25). The data were analyzed by a one-way analysis of variance followed by Duncan’s test. Statistical tests were performed using Dell Statistica 13 (Version 13.1 PL, Dell Inc., Tulsa, OK, USA). A value of p < 0.05 was considered significant.
experiment. Then, both groups were incubated in continuous darkness for two days. 2.5.3. Melatonin assay Melatonin concentration in the medium samples was measured by a direct RIA with the use of Prospect 6C antiserum (Paterson et al., 1992) and 3H-melatonin according to a previously described and validated procedure (Fraser et al., 1983; Prusik and Lewczuk, 2019). The sensitivity of the assay was 2.5 pg/tube. The intra- and interassay coefficients of variation were below 10%.
3. Results
2.5.4. Statistical analysis The data on the concentration of melatonin in medium samples from Experiment I were analyzed using repeated measures analysis of variance (ANOVA) with the treatment as the main effect and the sampling time as a repeated measure, separately for pineal organs of 10week-old and 40-week-old turkeys. The least significant difference test
3.1. Presence and distribution of pinopsin in the pineal organs The positive reaction with anti-pinopsin antibodies in the pineal organs of 2-day-old turkeys and 2-week-old turkeys was found mainly 5
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Fig. 5. The pineal organ of a forty-week-old turkey. A. Pinopsin immunoreactive structures. Note immunopositive apical prolongations of columnar cells (double arrows) and string-shaped or small spherical structures in the outer part of follicle wall (arrows). B. DAPI staining. The nuclei created several layers in the outer part of the follicle wall. C. Differential interference contrast. Note that the follicle wall is much thicker than in younger birds D. Imposition of figures A, B, C.
A prominent reduction in the number and size of pinopsin-immunopositive extensions protruding into the follicle lumen was noted in the pineal organs of 40- and 56-week-old turkeys (Fig. 5). Despite this, large amounts of amorphous pinopsin-positive material were present in the follicle lumen. Compared with younger turkeys, immunopositive string-shape or spherical structures were observed to be much more numerous in the outer part of the follicle wall. Additionally to the above described structures, weak immunoreactivity was also found in some cells scattered in the inner part of the follicle wall (Fig. 6).These cells were elongated with a bulbous protrusion that was in contact with the follicle lumen and a long basal process penetrating the outer part of the follicle wall.
in bulbous or multiform apical extensions of columnar cells protruding into the follicle lumen (Fig. 1). Sporadically, positive, fiber-like structures located between columnar cells, were observed (Fig. 2). In the pineal organs of 10-week-old turkeys, pinopsin immunoreactivity was also found mainly in the apical extensions of columnar cells (Fig. 3). Additionally, there were observed immunopositive fiber-like structures located between columnar cells and short string-shape structures in the outer part of the follicle wall. Two types of pinopsin-immunopositive structures were located in the follicle lumen in the pineal organs of 20-and 30-week-old turkeys (Fig. 4). The first type comprised apical extensions of columnar cells that were variable in shape and size. The second type was represented by amorphous material located in the central part of the follicle lumen. Several immunopositive short string-shape or small spherical structures were found in the outer part of the follicle wall. 6
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Fig. 6. Immunoreactive cell bodies (arrows) in the pineal organ of a two-week-old turkey (A, B) and a forty-week-old turkey (C, D). A., C. Pinopsin immunoreactive structures. B., D. Imposition of figure A with DAPI nuclear staining and differential interference contrast.
3.3. Direct photoreception in the regulation of melatonin secretion in the pineal organs of adult turkeys
3.2. Pinopsin localization in pinealocytes in monolayer culture The anti-pinopsin antibody staining was extremely intense in the short bulbous, conical or club-shaped processes growing out as a single structures from the cytoplasmic cell pole of pinealocytes (Fig. 7). The cell nucleus was usually located in the opposite pole of the pinealocyte, and one or sometimes two cell processes grew out from this part of the cell. Pinopsin-positive material was also observed in the cell bodies of some cells (Fig. 7). The double immunofluorescent staining with antipinopsin and anti-ASMT antibodies showed that pinopsin immunoreactivity was observed in almost all ASMT-positive cells (Fig. 8).
The pineal organs of 40-week-old turkeys incubated in a 12 L:12 D cycle secreted melatonin in a prominent diurnal rhythm (Figs. 9, 10). After the onset of darkness, the secretion increased stepwise to reach its maximum in the second half of scotophase and then slowly decreased. The lowest level of melatonin release was noted in the second half of photophase. The amplitude of the daily changes in melatonin release was lower in the pineal organs of 40-week-old turkeys than in those of 10-week-old birds. The circadian rhythm of melatonin secretion from the pineal organs of 40-week-old turkeys, such as those of 10-week-old birds, was maintained during two days of incubation in continuous darkness. 7
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4. Discussion In this study, we investigated the presence and distribution of pinopsin in the turkey pineal organ during post-hatching development. Immunohistochemical staining enabled us to identify four main types of pinopsin-containing structures in the turkey pineal organ. First, pinopsin immunoreactivity was found in the extensions protruding from the columnar cells into the follicle lumen. It is obvious that these structures correspond to the apical protrusions of rudimentary photoreceptor pinealocytes (Przybylska-Gornowicz et al., 2005; PetrusewiczKosińska et al., 2019). The inner part of the follicle wall contained immunopositive fiber-like structures located between columnar cells. They were especially numerous in the pineal organs of young turkeys; therefore, we speculate that they belong to developing rudimentary receptor pinealocytes. The third type of pinopsin immunoreactive structures comprises short string-shape or small spherical structures located in the outer part of the follicle wall. This part of the follicle is formed mainly by secretory pinealocytes (Petrusewicz-Kosińska et al., 2019), and it seems that immunopositive structures are processes of these cells. Immunohistochemistry also showed a positive material inside the follicle lumen. Based on our previous histological and ultrastructural studies and the data showing the long lifespan of excited pinopsin molecules (Nakamura et al., 1999), we have interpreted this material as cellular debris (Przybylska-Gornowicz et al., 2005; Petrusewicz-Kosińska et al., 2019). Its presence is important proof of the idea that the apical protrusions of rudimentary receptor pinealocytes undergo a specific renewal process with elimination of worn out parts of cells. Previous studies reported the presence of two types of pinopsinpositive structures in the avian pineal organs. An intense immunoreaction was found in the apical processes of rudimentary receptor pinealocytes in the pineal organ of the chicken (Hirunagi et al., 1997; Okano et al., 1997; Vigh et al., 1998; Matsushita et al., 2000), quail (Vigh et al., 1998; Yamao et al., 1999) and pigeon (Okano et al., 1997; Vigh et al., 1998). Pinopsin-positive string- or bulb-shaped elements were observed at the periphery of the follicles in the chicken pineal organ (Hirunagi et al., 1997; Okano et al., 1997). They were considered processes of parafollicular cells. Unlike the intensive pinopsin immunoreactivity observed in the apical extensions of rudimentary photoreceptor pinealocytes, their cell bodies were generally devoid of reaction products. These findings are in accordance with observations in chicken (Hirunagi et al., 1997; Okano et al., 1997; Matsushita et al., 2000), pigeon (Okano et al., 1997) and in the developing quail pineal organ (Yamao et al., 1999). The difference in the immunoreactivity of the apical extension and the cell body of pinealocytes was interpreted as the occurrence of the mechanism of pinopsin transport to specific cell sites that can function immediately after the onset of pinopsin synthesis (Yamao et al., 1999). Our observations confirm the existence of such a mechanism controlling the distribution of pinopsin in turkey pinealocytes. It is worth noting that in the inner part of the follicle wall, we found some cell bodies that were immunoreactive with anti-pinopsin antibodies. Their localization in the inner part of the follicle wall as well as their elongated shape and presence of immunopositive apical protrusions enables their identification as rudimentary receptor pinealocytes. Such pinopsin immunopositive cells have not been described thus far. We speculate that the presence of a detectable amount of pinopsin in a cell body is related to a specific pinealocyte development phase and is temporary. Transient disturbances in pinopsin transport to specific cell sites may be responsible for this phenomenon. The existing data on pinopsin immunoreactivity in the pineal organ concern almost exclusively young chickens from one to several days old (Takanaka et al., 1998; Holthues, 2005), two to four weeks old (Hirunagi et al., 1997; Nakahara et al., 1997) or two months old (Hirunagi et al., 1997; Okano et al., 1997). There are no studies on pinopsin in adult chickens. Hirunagi et al. (1997) reported no
Fig. 7. The positive staining for pinopsin in pinealocytes of turkey in monolayer culture (7 day of culture). The strong immunoreactivity is visible in short cytoplasmic processes outgrowing from the cytoplasmic pole of cells.
Fig. 8. The double immunofluorescent staining of turkey pinealocytes in the monolayer culture (7 days of culture) with antibodies against ASMT (red fluorescence) and pinopsin (green fluorescence). Take note of strong pinopsin immunoreactivity (green) in the short cytoplasmic processes (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Exposition to light during scotophase for 30 min or 60 min did not change the secretion of melatonin from the pineal organs of 40-weekold turkeys, while it induced a decrease in melatonin secretion and a shift of the endogenously generated rhythm in the organs of 10-weekold birds (Fig. 9). Light exposition for 120 min resulted in a decline in melatonin secretion and an advance of the endogenously generated rhythm in the pineal explants of both young and adult turkeys. The inhibitory effect of light exposition for two hours was stronger in birds aged 10 weeks than 40 weeks. The pineal organs of 40-week-old turkeys entrained the melatonin secretion rhythm to a reversed light-dark cycle starting from the second day of culture (Fig. 10). The reversed rhythm was also expressed during the incubation in continuous darkness.
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Fig. 9. Secretion of melatonin (mean) from the pineal organs of turkeys aged 10 (A) and 40 weeks (B) incubated under a 12 L:12 D cycle during the first two days of the experiment and then in continuous darkness for one day. The pineal organs were exposed to light (fluorescent lamp, 100 lx) starting at 01.00 for 30, 60 or 120 min during the second day of the experiment. Control explants were not exposed to light during scotophase. Values labeled with different letters differ significantly (p ≤ 0.05) between groups. * significant (p ≤ 0.05) shift of the midpoints of the peaks.
findings indicate that participation of secretory pinealocytes in the direct photosensitivity of the turkey pineal organ increases during development. In the next step of our research, we prepared a monolayer cell culture from pineal organs of twenty-week-old turkeys. At this phase of development, the organs comprise both types of pinealocytes (Petrusewicz-Kosińska et al., 2019), and we expected to have rudimentary photoreceptor and secretory pinealocytes in the culture. As mentioned in the Introduction, the aim of this part of study was to answer the question of whether the localization and morphology of pinopsin-positive structures in pinealocytes are determined by their contact with the follicle lumen or genetically programmed by a cell type. We used ASMT, the last enzyme in the melatonin synthesis pathway, as a marker of pinealocytes. Surprisingly, the morphology of ASMT-positive cells was much the same, which did not allow us to differentiate the two types of pinealocytes. All pinealocytes were polarized, more or less elongated and formed a single, short pinopsinpositive process outgrowing from the pinealocyte pole located opposite
remarkable age-dependent changes in pinopsin immunoreactivity in the pineal organ of chickens; however, this study covered only 1- to 2month-old young chickens. Concerning pinopsin in adult birds, only one study was performed on pigeons (Okano et al., 1997). Our study is the first one to investigate changes in pinopsin immunoreactivity in the avian pineal gland during post-hatching development from hatching to maturity. The obtained data provide clear evidence for the reduction of pinopsin immunoreactivity in rudimentary photoreceptor pinealocytes after sexual maturation of turkeys. This process overlaps with the observation of ultrastructure regressive changes of these cells, including reduction of the apical extension (Petrusewicz-Kosińska et al., 2019). It also coincides with the accumulation of immunoreactive cellular debris in the follicle lumen. On the other hand, the number of pinopsin immunopositive string-shape or spherical structures, located in the outer part of the follicle wall in the turkey pineal organ, prominently increases with age. This process correlates with a large increase in the number of secretory pinealocytes during postnatal development of the turkey pineal gland. These 9
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Fig. 10. Secretion of melatonin (mean) from the pineal organs of 40-week-old turkeys incubated under a 12 L:12 D cycle with the photophase from 07.00 to 19.00 or a reversed cycle of 12 D:12 L during three consecutive days and then in continuous darkness for two days.
12-week-old geese and 9-month-old ducks to a reversed dark-light cycle takes more than two days (Ohshima and Matsuo, 1984; Prusik et al., 2015; Ziółkowska, 2015). A change in the light-dark cycle also affects the circadian oscillator in pinealocytes of adult turkeys.
to the nucleus. Our data demonstrates that the mechanism responsible for pinopsin distribution acts in a single cell and is independent of the localization of the cell in the follicle. The results obtained in our experiment showed that all pinealocytes in the turkey pineal organ are photosensitive and showed apparent polarization in vitro. Taking into account the morphology of parafollicular cells in the chicken, it was suggested that these cells are modified photoreceptor pinealocytes with a more simplified structure than that of rudimentary photoreceptor cells (Ohshima and Matsuo, 1984; Hirunagi et al., 1997). Our data provide strong and direct evidence for the photoreceptor nature of secretory pinealocytes and support the above-mentioned suggestion. The changes in organization of the turkey pineal organ during postembryonic development offer the unique possibility of performing physiological studies on the glands, in which melatonin-synthetizing cells are represented almost exclusively by well-developed rudimentary receptor pinealocytes (in 4 – 12-week-old birds) or by regressed rudimentary receptor pinealocytes and highly numerous secretory cells (in 40–56 weeks old birds) (Przybylska-Gornowicz et al., 2005; Petrusewicz-Kosińska et al., 2019). In the present study, we compared the role of direct photoreception in the regulation of melatonin secretion in the organs of 10-week-old and 40-week-old turkeys. The obtained results show that the pineal organs of young birds are much more sensitive to light exposition at night than the pineal organs of adult turkeys. Short light exposition during scotophase induced a decrease in melatonin secretion in the pineal organs of young turkeys but not in adult birds. These data demonstrate, for the first time, functional differences between the two kinds of pinopsin-containing cells: rudimentary receptor pinealocytes with an internal organization resembling retinal photoreceptors are more light-sensitive than secretory pinealocytes resembling neurons. It is worth noting that the pineal organs of adult turkeys secrete melatonin in a well-entrained diurnal rhythm, with slightly lower amplitude than the glands of turkeys 10–14 weeks of age (Prusik and Lewczuk, 2019). For comparison, entrainment to a light-dark cycle is much weaker in the pineal organs of young ducks (Prusik et al., 2015), and especially in young geese (Ziółkowska, 2015), than in adult turkeys. It should also be underlined that despite the lower light sensitivity, the pineal organs of adult turkeys adopt their secretory activity to the reversed light-dark cycle during the second day of culture in 12 D:12 L conditions, as do the pineal organs of young turkeys. Such adaptation in the pineal organ pinealocytes of 15-week-old chickens,
5. Conclusions Our results showed that pinopsin is present in the turkey pineal organ during the entire period of postnatal development, from hatching to maturity; however, its distribution changes during this time. Agedependent reduction of pinopsin immunoreactive apical extensions of rudimentary photoreceptor pinealocytes occurs together with a simultaneous increase in pinopsin-immunopositive elements in the outer part of the follicle wall, composed of secretory pinealocytes. All pinealocytes in the turkey pineal organ are photoreceptive; however, secretory pinealocytes are less light-sensitive than rudimentary receptor pinealocytes. The autonomous regulatory mechanisms, including direct photoreception and endogenous rhythm generation, work well in the pineal organs of adult turkeys. Funding This study was founded by KNOW (Leading National Research Centre) Scientific Consortium “Healthy Animal – Safe Food” decision of Ministry of Science And Higher Education no. 05-1/KNOW2/2015. The publication costs were supported by the Minister of Science and Higher Education under the program entitled "Regional Initiative of Excellence" for the years 2019–2022, Project No. 010/RID/2018/19; the amount of funding was 12.000.000 PLN. Declaration of Competing Interest The authors declare that they have no conflicts of interest. Acknowledgments The authors wish to express their deep thanks to Professor Yoshitaka Fukada and Professor Toshiyuki Okano from The University of Tokyo, Japan for the gift of pinopsin antiserum. The authors also would like to thank Dr Andrew Foldes from the Division of Animal Production, CSIRO, Blacktown, Australia for the gift of anti-melatonin antibodies. 10
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The authors wish to express their deep thanks to Dr Pierre Voisin from The University in Poitiers, France for the gift of ASMT antiserum.
molecule. Nature 372, 94–97. Okano, T., Fukada, Y., 1997. Phototransduction cascade and circadian oscillator in chicken pineal gland. J. Pineal Res. 22, 145–151. Okano, T., Takanaka, Y., Nakamura, A., Hirunagi, K., Adachi, A., Ebihar, S., Fukada, Y., 1997. Immunocytochemical identification of pinopsin in pineal glands of chicken and pigeons. Brain Res. Mol. Brain Res. 50, 190–196. Paterson, A.M., Martin, G.B., Foldes, A., Maxwell, C.A., Pearce, G.P., 1992. Concentrations of plasma melatonin and luteinizing hormone in domestic gilts reared under artificial long and short days. J. Reprod. Fertil. 94, 85–95. Petrusewicz-Kosińska, M., Przybylska-Gornowicz, B., Ziółkowska, N., Martyniuk, K., Lewczuk, B., 2019. Developmental morphology of the turkey pineal organ. Immunocytochemical and ultrastructural studies. Micron 122, 8–20. Prusik, M., Lewczuk, B., Ziółkowska, N., Przybylska-Gornowicz, B., 2015. Regulation of melatonin seretion in the pineal organ of the domestic duck – an in vitro study. Pol. J. Vet. Sci. 18, 635–644. Prusik, M., Lewczuk, B., 2019. Roles of direct photoreception and the internal circadian oscillator in the regulation of melatonin secretion in the pineal organ of the domestic turkey. A novel in vitro clock and calendar model. Int. J. Mol. Sci. 20, 4022. Przybylska-Gornowicz, B., Lewczuk, B., Prusik, M., Nowicki, M., 2005. Post-hatching development of the turkey pineal organ: histological and immunohistochemical studies. Neuro Endocrinol. Lett. 26, 383–392. Quay, W.B., Renzoni, A., 1967. The diencephalic relations and variability in the double structure of the epiphyseal complex of birds. Riv. Biol. 60, 9–75. Sato, T., Wake, K., Kramm, C., Korf, H.W., 1990. Chicken pineal organs during posthatching development: photoreceptor-specific characteristics and innervation. In: Gupta, D., Wollmann, H., Ranke, B. (Eds.), Neuroendocrinology: New Frontiers. Research Promotion, Tubingen, pp. 191–200. Sato, T., 2001. Sensory and endocrine characteristics of the avian pineal organ. Microsc. Res. Tech. 53, 2–11. Takanaka, Y., Okano, T., Iigo, M., Fukada, Y., 1998. Light-dependent expression of pinopsin gene in chicken pineal gland. J. Neurochem. 70, 908–913. Vigh, B., Röhlich, P., Görcs, T., Manzano e Silva, M.J., Szél, A., Fejér, Z., Vígh-Teichmann, I., 1998. The pineal organ as a folded retina: immunocytochemical localization of opsins. Biol. Cell 90, 653–659. Voisin, P., Guerlotté, J., Collin, J.P., 1988. An antiserum against chicken hydroxyindoleO-methyltransferase reacts with the enzyme from pineal gland and retina and labels pineal modified photoreceptors. Brain Res. 464, 53–61. Vollrath, L., 1981. The Pineal Organ. Handbuch Der Mikroskopischen Anatomie Des Menschen VI/7. Springer-Verlag, Berlin, pp. 437–458. Yamao, M., Araki, M., Okano, T., Fukada, Y., Oishi, T., 1999. Differentiation of pinopsinimmunoreactive cells in the developing quail pineal organ: an in-vivo and in-vitro immunohistochemical study. Cell Tissue Res. 296, 667–671. Ziółkowska, N., 2015. Melatonin Biosynthesis and Mechanisms of Its Regulation in the Domestic Goose Pineal Organ. Doctoral thesis. University of Warmia and Mazury, Olsztyn, Poland.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.micron.2019.102749. References Bertolucci, C., Foa, A., 2004. Extraocular photoreception and circadian entrainment in nonmammalian vertebrates. Chronobiol. Int. 21, 501–519. Collin, J.P., Falcon, J., Voisin, P., Brisson, P., 1986. The pineal organ: ontogenic differentiation of photoreceptor cells and pinealocytes. In: Gupta, D., Reiter, R.J. (Eds.), The Pineal Gland During Development: From Fetus to Adult. Croom Helm, London & Sydney, pp. 14–30. Fraser, S., Cowen, P., Franklin, M., Franey, C., Arendt, J., 1983. Direct radioimmunoassay for melatonin in plasma. Clin. Chem. 29, 396–397. Hirunagi, K., Ebihara, S., Okano, T., Takanaka, Y., Fukada, Y., 1997. Immunoelectronmicroscopic investigation of the subcellular localization of pinopsin in the pineal organ of the chicken. Cell Tissue Res. 289, 235–241. Holthues, H., Engel, L., Spessert, R., Vollrath, L., 2005. Circadian gene expression patterns of melanopsin and pinopsin in the chick pineal gland. Biochem. Biophys. Res. Commun. 326, 160–165. Kasahara, T., Okano, T., Yoshikawa, T., Yamazaki, K., Fukada, Y., 2000. Rod-type transducin alpha-subunit mediates a phototransduction pathway in the chicken pineal gland. J. Neurochem. 75, 217–224. Mano, H., Fukada, Y., 2007. A median third eye: pineal gland retraces evolution of vertebrate photoreceptive organs. Photochem. Photobiol. 83, 11–18. Matsushita, A., Yoshikawa, T., Okano, T., Kasahara, T., Fukada, Y., 2000. Colocalization of pinopsin with two types of G-protein alpha-subunits in the chicken pineal gland. Cell Tissue Res. 299, 245–251. Nakahara, K., Murakami, N., Nasu, T., Kuroda, H., Murakami, T., 1997. Individual pineal cells in chick possess photoreceptive, circadian clock and melatonin-synthesizing capacities in vitro. Brain Res. 774, 242–245. Nakamura, A., Kojima, D., Imai, H., Terakita, A., Okano, T., Shichida, Y., Fukada, Y., 1999. Chimeric nature of pinopsin between rod and cone visual pigments. Biochem 38, 14738–14745. Ohshima, K., Matsuo, S., 1984. Functional morphology of the pineal gland in young chickens. Anat. Anz. Jena. 156, 407–418. Ohshima, K., Hiramatsu, K., 1993. Ultrastructural study of post-hatching development in the pineal gland of the Japanese quail. J. Vet. Med. Sci. 55, 945–950. Okano, T., Yoshizawa, T., Fukada, Y., 1994. Pinopsin is a chicken pineal photoreceptive
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Pinopsin and photoreception in the pineal organ of the domestic turkey during post-hatching development
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Marcela Petrusewicz-Kosińska, Barbara Przybylska-Gornowicz , Magdalena Prusik, Natalia Ziółkowska, Bogdan Lewczuk Department of Histology and Embryology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego 13, 10-719 Olsztyn, Poland
A R T I C LE I N FO
A B S T R A C T
Keywords: Pineal organ Extraocular photoreception Pinopsin Melatonin Development Birds
The avian pineal organ is photosensitive because of the presence of photopigments, of which pinopsin seems to be one of the most important. This organ is subject to far-reaching changes during post-hatching development, but evidence regarding pinopsin presence and direct photoreception during this time is lacking. This study was carried out to demonstrate the following: 1) the structures showing immunoreactivity to pinopsin in the turkey pineal organ, 2) the changes of these structures during development, 3) the pinopsin localization in pinealocytes in monolayer cultures, and 4) the role of direct photoreception in the regulation of melatonin secretion in pineal organs in adult turkeys. Pinopsin immunoreactivity was localized in the apical extensions of columnar cells limiting the follicular lumen, in fiber-like structures located between columnar cells in the inner part of follicle wall, in string-shapes or small spherical structures distributed in the outer part of follicle wall and in amorphous material inside the follicle lumen. In young birds, immunoreactivity was also sporadically noted in cell bodies of rudimentary receptor pinealocytes. The distribution of pinopsin showed prominent age-dependent changes, including a subsequent increase in pinopsin-positive structures in the outer part of the follicle wall and a prominent reduction in the number and size of positive apical extensions in 40- and 56-week-old turkeys. These data demonstrate that the role of secretory pinealocytes in pineal photoreception increases with age. In monolayer cultures, all pinealocytes showed strong reactions in club- or bulbous-shaped prolongations. The pineal organs of adult birds were less sensitive to light exposition at night than those of young turkeys, which points to differences in light sensitivity between rudimentary receptor and secretory pinealocytes. However, direct photoreception could play an important role in the regulation of melatonin secretion in adult turkeys.
1. Introduction Avian pinealocytes possess photoreceptive capability, endogenous circadian oscillator and melatonin synthesizing machinery (Nakahara et al., 1997). The predominant pineal photoreceptive molecule in birds, pinopsin, controls the daily melatonin output (Takanaka et al., 1998; Holthues et al., 2005; Mano and Fukada, 2007). Pinopsin belongs to the nonvisual opsins, which are detected in extraocular photoreceptive sites localized in the pineal complex and other structures of the brain in nonmammalian vertebrates (Bertolucci and Foa, 2004). Pinopsin was initially cloned from the chicken pineal organ (Okano et al., 1994), and its expression is probably limited to birds. Pinopsin possesses a rhodopsin-like heptahelical primary structure with a chromophore of 11-cis-retinal (Okano and Fukada, 1997) and exhibits sensitivity to blue light, with maximum absorbance at
approximately 470 nm (Okano et al., 1994; Nakamura et al., 1999). It is a member of the GTP-binding protein-coupled receptor family, and the light signal captured by pinopsin is most likely transduced by heterotrimeric G-protein. The colocalization of G-protein α-subunits, the rod transducin Gt1α and Gq/11α, with pinopsin suggests that these subunits are functionally coupled with this opsin (Matsushita et al., 2000). Although the precise role of pinopsin has not been finally elucidated, there is evidence of its mediation of the acute effect of light on melatonin secretion by the Gt1α pathway (Kasahara et al., 2000). Moreover, colocalization of Gq/11α with pinopsin suggests that this photopigment is also involved in entrainment of the intrinsic circadian oscillator (Matsushita et al., 2000; Mano and Fukada, 2007). To date, the presence and localization of pinopsin in the pineal organ of birds have been studied only fragmentarily in a few species, such as young chicken (Hirunagi et al., 1997; Okano et al., 1997), adult
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Corresponding author. E-mail addresses: [email protected] (M. Petrusewicz-Kosińska), [email protected] (B. Przybylska-Gornowicz), [email protected] (M. Prusik), [email protected] (N. Ziółkowska), [email protected] (B. Lewczuk). https://doi.org/10.1016/j.micron.2019.102749 Received 2 August 2019; Received in revised form 2 September 2019; Accepted 2 September 2019 Available online 03 September 2019 0968-4328/ © 2019 Elsevier Ltd. All rights reserved.
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were anesthetized with isoflurane and killed by decapitation between 08:00 and 09:00 at the age of 2 days, 2, 10, 20, 30, 40 and 56 weeks. The pineal organs with adjacent parts of the brain were immediately removed and prepared for immunohistochemical study. Additionally, 10 pineal organs of 20-week-old turkeys were collected for the preparation of a monolayer cell culture, and 30 pineal organs were obtained from birds at the ages of 10 weeks (n = 12) and 40 weeks (n = 18) for a superfusion culture. All experimental procedures involving animals were carried out in compliance with Polish legal regulations determining the terms and methods of performing experiments on animals.
pigeons (Okano et al., 1997) and quail embryos (Yamao et al., 1999). Pinopsin immunoreactivity has been found in the pear-shaped apical protrusions of follicular cells extending into the follicle lumen (Hirunagi et al., 1997; Okano et al., 1997; Yamao et al., 1999) and in long slender structures in the parafollicular pineal parenchyma (Hirunagi et al., 1997). The avian pineal organs vary considerably in their anatomical forms (Quay and Renzoni, 1967; Vollrath, 1981) and histological organization (Vollrath, 1981; Ohshima and Matsuo, 1984; Sato, 2001). The structure of this organ also undergoes prominent changes during ontogenesis (Sato et al., 1990; Ohshima and Hiramatsu, 1993; Sato, 2001; Przybylska-Gornowicz et al., 2005; Petrusewicz-Kosińska et al., 2019). It should be stressed that comparative studies on the presence and distribution of pinopsin are still lacking, and as a consequence, very little is known about the localization of this photomolecule in different forms of the avian pineal organ. Literally nothing is known about changes in the distribution of this photomolecule during the posthatching transformation of the pineal organ. In our previous studies, we have traced morphological aspects of developmental processes of the domestic turkey pineal organ during the period from one day until one year of post-hatching life (PrzybylskaGornowicz et al., 2005; Petrusewicz-Kosińska et al., 2019). The turkey pineal organ retains a follicular structure throughout the entire investigated period and comprises two types of pinealocytes, rudimentary photoreceptor pinealocytes and secretory pinealocytes. Rudimentary photoreceptor pinealocytes compose a part of the follicle wall surrounding the lumen and develop until the age of 20 weeks. This process leads to the appearance of extremely well-developed cells with prominent apical prolongation and stratified organelle distribution in turkeys at the ages of 20 and 30 weeks. The stratified distribution of organelles is similar to the compartmentalization of retinal photoreceptors and pineal photoreceptors of lower vertebrates, which are involved in phototransduction (Collin et al., 1986). Then, rudimentary photoreceptor cells show pronounced regressive changes in 40- and 56-weekold birds; the number and size of the organelles are distinctly reduced, and apical protrusions are reduced. In contrast to rudimentary photoreceptor pinealocytes, secretory pinealocytes lack contact with the follicle lumen and polarization. Their number increases during the postnatal development, and they form a prominent outer part of the follicle wall in 40- and 56-week-old turkeys (Petrusewicz-Kosińska et al., 2019). The changes in the organization of the follicles suggest the reduction of photoreceptor capability of the turkey pineal after sexual maturation. The results of morphological studies prompted us to ask the question of whether the observed changes also apply to pinopsin, the main sensory input system of the avian pineal organ. In this study, we used immunochemical staining to examine the presence and distribution of pinopsin in the pineal organs of turkeys at various stages of posthatching life. We also prepared a monolayer culture of turkey pineal cells to check whether the localization and morphology of pinopsinpositive structures in pinealocytes is determined by their contact with the follicle lumen or if it is genetically programmed by the type of cell (rudimentary receptor or secretory pinealocyte). Finally, we compared in vitro the effect of light exposure during scotophase on melatonin secretion from the pineal organs of 10-week-old and 40-week-old turkeys as well as their ability to entrain secretory activity to a reversed light-dark cycle.
2.2. Chemicals The mouse monoclonal antibody against pinopsin was obtained from Prof. Y. Fukada and Prof. T. Okano from the University of Tokyo, Japan. A procedure for preparation of the antibody as well as validation of its specificity was described in detail by Okano et al. (1997). The rabbit primary antiserum against chicken N-acetylserotonin O-methyltransferase (ASMT) was a gift of Dr Pierre Voisin, University of Poitiers, France. A procedure for preparation and characterization of the antibody as well as validation of its specificity was described in detail by Voisin et al. (1988). Secondary antibodies coupled with AlexaFluor 488 or AlexaFluor 594 and DAPI were purchased from Molecular Probes (Eugene, OR, USA). The anti-melatonin antibody Prospect 6C was kindly provided by Dr Andrew Foldes (Division of Animal Production, CSIRO, Blacktown, Australia). 3H-melatonin was purchased from PerkinElmer (Waltham, MA, USA). Gelatin was obtained from Merck (Billerica, MA, USA) and paraformaldehyde from Polysciences, Inc. (Warrington, PA, USA). Medium 199 with Earle’s salt and HEPES, Hank’s balanced sodium salt, Earle’s balanced sodium salt, Dulbecco's modified eagle medium, antibiotic-antimycotic solution, fetal bovine serum, collagenase IV, DNA-se, poly-L-lysine and all other chemicals were purchased from Sigma (St. Louis, MO, USA). 2.3. Immunohistochemical studies The pineal organs were fixed in a 4% paraformaldehyde solution in 0.1 M phosphate buffer (pH = 7.4) for 45 min. Then, the tissues were rinsed with phosphate buffer, cryoprotected in 25% sucrose solution, frozen and cut into 10 μm thick sections using a Microm HM 560 cryostat (Microm, Walldorf, Germany). The sections mounted on glass slides were subjected to an immunohistochemical procedure using the mouse monoclonal antibody against pinopsin (working dilution of 5 μg/ ml). The primary antibodies bound to the antigens present in the tissue slides were visualized by means of secondary goat anti-mouse IgG antiserum coupled with AlexaFluor 488. After intense washing, cell nuclei were stained in DAPI solution, and the sections were sealed in a mixture of polyvinyl alcohol and glycerine with 2% DABCO. The sections were examined using a fluorescence microscope equipped with a digital camera (Carl Zeiss, Oberkochen, Germany). The control procedures were performed with omission of the primary antisera. 2.4. Monolayer cell culture and immunocytochemistry 2.4.1. Cell culture The pineal organs of 20-week-old turkeys were collected in Hank’s Balanced Sodium Salt containing penicillin (1000 Ul/ml), streptomycin (1 mg/ml) and amphotericin B (2.5 μg/ml) and transported to the cell culture laboratory within 10 min after the animals’ death. The organs were cut into several parts and subjected to enzymatic digestion performed at 37 °C for 60 min in a solution containing collagenase IV (2 mg/ml) and DNase (4 μg/ml) in Earle’s balanced sodium salt. After dispersion, the cells were washed three times with culture medium containing 10% bovine fetal serum and then suspended in Dulbecco's Modified Eagle Medium with fetal calf serum (10%), penicillin (100 Ul/
2. Materials and methods 2.1. Animals and pineal glands The study was performed on females of the domestic turkey (Meleagris gallopavo). The birds (apart from those that were newly hatched) were housed under a cycle consisting of 12 h of light and 12 h of dark (light from 07:00 to 19:00). The turkeys (six for each age group) 2
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Fig. 1. Immunohistochemical localization of pinopsin in the pineal organ of two-day-old turkey. A. Pinopsin immunoreactive structures. B. DAPI nuclear staining; C. Differential interference contrast; D – imposition of figures A, B, C. Note presence of positive reaction exclusively in the apical protrusions extending into the follicle lumen (A), the regular arrangement of cell nuclei (B) and the shape of cells (C, D).
ml), streptomycin (0.1 mg/ml) and amphotericin B (0.25 μg/ml). The cells were placed in culture plates covered by poly-L-lysine. Culture was performed at 38 °C under a 12 h light:12 h dark cycle (12 L:12 D) with fluorescent illumination between 09:00 and 21:00. The medium was changed 48 h after the plating and then every 12 h. Cell morphology during the culture was investigated using inverted microscope equipment with phase and relief contrasts (Carl Zeiss, Oberkochen, Germany).
secondary goat anti-mouse IgG antiserum coupled with AlexaFluor 488 and the anti-rabbit IgG coupled with Alexa 594. The control procedures were performed with omission of the primary antisera. The sections were examined using an inverted fluorescence microscope equipped with a digital camera (Carl Zeiss Microscopy, Oberkochen, Germany). 2.5. Superfusion culture and melatonin secretion study 2.5.1. Superfusion culture The pineal organs were collected in Medium 199 containing penicillin (100 Ul/ml), streptomycin (0.1 mg/ml) and amphotericin B (0.25 μg/ml) and transported to the cell culture laboratory within 10 min after the animals’ death. Next, the pineal organs were covered with nylon mesh and placed in culture chambers. The lower pool of each chamber was connected to the vessel with culture medium. The
2.4.2. Immunocytochemistry After 7 days, the cell cultures were fixed with 4% paraformaldehyde for 15 min and subjected to a double immunohistochemical procedure using mouse monoclonal antiserum against pinopsin (5 μg/ml) and rabbit primary antiserum against chicken ASMT (working dilution 1:500). The antigen-antibody complexes were revealed by the 3
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Fig. 2. Immunohistochemical localization of pinopsin in the pineal organ of two-week-old turkey. A. Pinopsin immunoreactive structures. B. Imposition of figure A with DAPI nuclear staining. Note presence of fiber-like structures located between columnar cells in the inner part of the follicle wall.
Fig. 3. Immunohistochemical localization of pinopsin in the pineal organ of ten-week-old turkey. A. Pinopsin immunoreactive structures. B. Imposition of figure A with DAPI nuclear staining and differential interference contrast. Note presence of positive structures in the outer part of the follicle wall (arrows).
2.5.2. Experimental procedures Two experiments were performed. In experiment I, pineal organs obtained from 10-week-old and 40-week-old turkeys were incubated under a 12 L:12 D cycle during the first two days of the experiment and then in continuous darkness for two days. The explants of both age groups were exposed to light (fluorescent lamp, 100 lx) beginning at 01.00 for 30 (n = 3), 60 (n = 3) or 120 (n = 3) minutes during the second day of the experiment. The control pineal organs (n = 3) were not exposed to the light during scotophase. The second experiment was performed on two groups of the pineal organs collected from 40-week-old turkeys. The first group (n = 3) was incubated under a 12 L:12 D cycle and the second group (n = 3) under a reversed cycle of 12 D:12 L during the first three days of the
upper pool of the culture chamber was attached to a multichannel peristaltic pump (Cole Parmer, Vernon Hills, IL, USA) and a manual fraction collector. The chamber was superfused at a rate of 0.1 ml/min with Medium 199 containing penicillin (100 Ul/ml), streptomycin (0.1 mg/ml) and amphotericin B (0.25 μg/ml), which was continuously gassed with a mixture of 95% O2 and 5% CO2. The incubation was performed at 38 °C in a water bath (Julabo, Seelbach, Germany). The chambers were illuminated with a full-spectrum fluorescent lamp providing light with an intensity of 100 lx at their surface. They were covered with light-proof plastic sheets during scotophase and with similar translucent sheets during incubation during photophase. Medium samples were collected every 30 min and frozen at −20 °C until melatonin assays were carried out. 4
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Fig. 4. The pineal organ of a twenty-week-old turkey. A. Pinopsin immunoreactive structures. B. DAPI staining. C. Differential interference contrast, D. Imposition of figures A, B, C. Note strong immunoreactivity situated in amorphous material in the lumen of follicle (star), in the apical prolongations of columnar cells (double arrow) and in string-shaped or small spherical structures in the outer part of follicle wall (arrows). The nuclei created a compact layer in the inner part of the follicle wall and were loosely dispersed in the outer part.
(LSD) was applied as a post hoc procedure. To assess the changes in a course of the melatonin secretion rhythm, the middle of each peak was determined as the point (daily time) halfway between the point at which secretion increased to 75% of the maximum value and the point at which secretion decreased to 75% of the maximum value. To perform statistical analysis, minutes were expressed as tenths of hours (e.g., 15:15 = 15.25). The data were analyzed by a one-way analysis of variance followed by Duncan’s test. Statistical tests were performed using Dell Statistica 13 (Version 13.1 PL, Dell Inc., Tulsa, OK, USA). A value of p < 0.05 was considered significant.
experiment. Then, both groups were incubated in continuous darkness for two days. 2.5.3. Melatonin assay Melatonin concentration in the medium samples was measured by a direct RIA with the use of Prospect 6C antiserum (Paterson et al., 1992) and 3H-melatonin according to a previously described and validated procedure (Fraser et al., 1983; Prusik and Lewczuk, 2019). The sensitivity of the assay was 2.5 pg/tube. The intra- and interassay coefficients of variation were below 10%.
3. Results
2.5.4. Statistical analysis The data on the concentration of melatonin in medium samples from Experiment I were analyzed using repeated measures analysis of variance (ANOVA) with the treatment as the main effect and the sampling time as a repeated measure, separately for pineal organs of 10week-old and 40-week-old turkeys. The least significant difference test
3.1. Presence and distribution of pinopsin in the pineal organs The positive reaction with anti-pinopsin antibodies in the pineal organs of 2-day-old turkeys and 2-week-old turkeys was found mainly 5
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Fig. 5. The pineal organ of a forty-week-old turkey. A. Pinopsin immunoreactive structures. Note immunopositive apical prolongations of columnar cells (double arrows) and string-shaped or small spherical structures in the outer part of follicle wall (arrows). B. DAPI staining. The nuclei created several layers in the outer part of the follicle wall. C. Differential interference contrast. Note that the follicle wall is much thicker than in younger birds D. Imposition of figures A, B, C.
A prominent reduction in the number and size of pinopsin-immunopositive extensions protruding into the follicle lumen was noted in the pineal organs of 40- and 56-week-old turkeys (Fig. 5). Despite this, large amounts of amorphous pinopsin-positive material were present in the follicle lumen. Compared with younger turkeys, immunopositive string-shape or spherical structures were observed to be much more numerous in the outer part of the follicle wall. Additionally to the above described structures, weak immunoreactivity was also found in some cells scattered in the inner part of the follicle wall (Fig. 6).These cells were elongated with a bulbous protrusion that was in contact with the follicle lumen and a long basal process penetrating the outer part of the follicle wall.
in bulbous or multiform apical extensions of columnar cells protruding into the follicle lumen (Fig. 1). Sporadically, positive, fiber-like structures located between columnar cells, were observed (Fig. 2). In the pineal organs of 10-week-old turkeys, pinopsin immunoreactivity was also found mainly in the apical extensions of columnar cells (Fig. 3). Additionally, there were observed immunopositive fiber-like structures located between columnar cells and short string-shape structures in the outer part of the follicle wall. Two types of pinopsin-immunopositive structures were located in the follicle lumen in the pineal organs of 20-and 30-week-old turkeys (Fig. 4). The first type comprised apical extensions of columnar cells that were variable in shape and size. The second type was represented by amorphous material located in the central part of the follicle lumen. Several immunopositive short string-shape or small spherical structures were found in the outer part of the follicle wall. 6
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Fig. 6. Immunoreactive cell bodies (arrows) in the pineal organ of a two-week-old turkey (A, B) and a forty-week-old turkey (C, D). A., C. Pinopsin immunoreactive structures. B., D. Imposition of figure A with DAPI nuclear staining and differential interference contrast.
3.3. Direct photoreception in the regulation of melatonin secretion in the pineal organs of adult turkeys
3.2. Pinopsin localization in pinealocytes in monolayer culture The anti-pinopsin antibody staining was extremely intense in the short bulbous, conical or club-shaped processes growing out as a single structures from the cytoplasmic cell pole of pinealocytes (Fig. 7). The cell nucleus was usually located in the opposite pole of the pinealocyte, and one or sometimes two cell processes grew out from this part of the cell. Pinopsin-positive material was also observed in the cell bodies of some cells (Fig. 7). The double immunofluorescent staining with antipinopsin and anti-ASMT antibodies showed that pinopsin immunoreactivity was observed in almost all ASMT-positive cells (Fig. 8).
The pineal organs of 40-week-old turkeys incubated in a 12 L:12 D cycle secreted melatonin in a prominent diurnal rhythm (Figs. 9, 10). After the onset of darkness, the secretion increased stepwise to reach its maximum in the second half of scotophase and then slowly decreased. The lowest level of melatonin release was noted in the second half of photophase. The amplitude of the daily changes in melatonin release was lower in the pineal organs of 40-week-old turkeys than in those of 10-week-old birds. The circadian rhythm of melatonin secretion from the pineal organs of 40-week-old turkeys, such as those of 10-week-old birds, was maintained during two days of incubation in continuous darkness. 7
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4. Discussion In this study, we investigated the presence and distribution of pinopsin in the turkey pineal organ during post-hatching development. Immunohistochemical staining enabled us to identify four main types of pinopsin-containing structures in the turkey pineal organ. First, pinopsin immunoreactivity was found in the extensions protruding from the columnar cells into the follicle lumen. It is obvious that these structures correspond to the apical protrusions of rudimentary photoreceptor pinealocytes (Przybylska-Gornowicz et al., 2005; PetrusewiczKosińska et al., 2019). The inner part of the follicle wall contained immunopositive fiber-like structures located between columnar cells. They were especially numerous in the pineal organs of young turkeys; therefore, we speculate that they belong to developing rudimentary receptor pinealocytes. The third type of pinopsin immunoreactive structures comprises short string-shape or small spherical structures located in the outer part of the follicle wall. This part of the follicle is formed mainly by secretory pinealocytes (Petrusewicz-Kosińska et al., 2019), and it seems that immunopositive structures are processes of these cells. Immunohistochemistry also showed a positive material inside the follicle lumen. Based on our previous histological and ultrastructural studies and the data showing the long lifespan of excited pinopsin molecules (Nakamura et al., 1999), we have interpreted this material as cellular debris (Przybylska-Gornowicz et al., 2005; Petrusewicz-Kosińska et al., 2019). Its presence is important proof of the idea that the apical protrusions of rudimentary receptor pinealocytes undergo a specific renewal process with elimination of worn out parts of cells. Previous studies reported the presence of two types of pinopsinpositive structures in the avian pineal organs. An intense immunoreaction was found in the apical processes of rudimentary receptor pinealocytes in the pineal organ of the chicken (Hirunagi et al., 1997; Okano et al., 1997; Vigh et al., 1998; Matsushita et al., 2000), quail (Vigh et al., 1998; Yamao et al., 1999) and pigeon (Okano et al., 1997; Vigh et al., 1998). Pinopsin-positive string- or bulb-shaped elements were observed at the periphery of the follicles in the chicken pineal organ (Hirunagi et al., 1997; Okano et al., 1997). They were considered processes of parafollicular cells. Unlike the intensive pinopsin immunoreactivity observed in the apical extensions of rudimentary photoreceptor pinealocytes, their cell bodies were generally devoid of reaction products. These findings are in accordance with observations in chicken (Hirunagi et al., 1997; Okano et al., 1997; Matsushita et al., 2000), pigeon (Okano et al., 1997) and in the developing quail pineal organ (Yamao et al., 1999). The difference in the immunoreactivity of the apical extension and the cell body of pinealocytes was interpreted as the occurrence of the mechanism of pinopsin transport to specific cell sites that can function immediately after the onset of pinopsin synthesis (Yamao et al., 1999). Our observations confirm the existence of such a mechanism controlling the distribution of pinopsin in turkey pinealocytes. It is worth noting that in the inner part of the follicle wall, we found some cell bodies that were immunoreactive with anti-pinopsin antibodies. Their localization in the inner part of the follicle wall as well as their elongated shape and presence of immunopositive apical protrusions enables their identification as rudimentary receptor pinealocytes. Such pinopsin immunopositive cells have not been described thus far. We speculate that the presence of a detectable amount of pinopsin in a cell body is related to a specific pinealocyte development phase and is temporary. Transient disturbances in pinopsin transport to specific cell sites may be responsible for this phenomenon. The existing data on pinopsin immunoreactivity in the pineal organ concern almost exclusively young chickens from one to several days old (Takanaka et al., 1998; Holthues, 2005), two to four weeks old (Hirunagi et al., 1997; Nakahara et al., 1997) or two months old (Hirunagi et al., 1997; Okano et al., 1997). There are no studies on pinopsin in adult chickens. Hirunagi et al. (1997) reported no
Fig. 7. The positive staining for pinopsin in pinealocytes of turkey in monolayer culture (7 day of culture). The strong immunoreactivity is visible in short cytoplasmic processes outgrowing from the cytoplasmic pole of cells.
Fig. 8. The double immunofluorescent staining of turkey pinealocytes in the monolayer culture (7 days of culture) with antibodies against ASMT (red fluorescence) and pinopsin (green fluorescence). Take note of strong pinopsin immunoreactivity (green) in the short cytoplasmic processes (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Exposition to light during scotophase for 30 min or 60 min did not change the secretion of melatonin from the pineal organs of 40-weekold turkeys, while it induced a decrease in melatonin secretion and a shift of the endogenously generated rhythm in the organs of 10-weekold birds (Fig. 9). Light exposition for 120 min resulted in a decline in melatonin secretion and an advance of the endogenously generated rhythm in the pineal explants of both young and adult turkeys. The inhibitory effect of light exposition for two hours was stronger in birds aged 10 weeks than 40 weeks. The pineal organs of 40-week-old turkeys entrained the melatonin secretion rhythm to a reversed light-dark cycle starting from the second day of culture (Fig. 10). The reversed rhythm was also expressed during the incubation in continuous darkness.
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Fig. 9. Secretion of melatonin (mean) from the pineal organs of turkeys aged 10 (A) and 40 weeks (B) incubated under a 12 L:12 D cycle during the first two days of the experiment and then in continuous darkness for one day. The pineal organs were exposed to light (fluorescent lamp, 100 lx) starting at 01.00 for 30, 60 or 120 min during the second day of the experiment. Control explants were not exposed to light during scotophase. Values labeled with different letters differ significantly (p ≤ 0.05) between groups. * significant (p ≤ 0.05) shift of the midpoints of the peaks.
findings indicate that participation of secretory pinealocytes in the direct photosensitivity of the turkey pineal organ increases during development. In the next step of our research, we prepared a monolayer cell culture from pineal organs of twenty-week-old turkeys. At this phase of development, the organs comprise both types of pinealocytes (Petrusewicz-Kosińska et al., 2019), and we expected to have rudimentary photoreceptor and secretory pinealocytes in the culture. As mentioned in the Introduction, the aim of this part of study was to answer the question of whether the localization and morphology of pinopsin-positive structures in pinealocytes are determined by their contact with the follicle lumen or genetically programmed by a cell type. We used ASMT, the last enzyme in the melatonin synthesis pathway, as a marker of pinealocytes. Surprisingly, the morphology of ASMT-positive cells was much the same, which did not allow us to differentiate the two types of pinealocytes. All pinealocytes were polarized, more or less elongated and formed a single, short pinopsinpositive process outgrowing from the pinealocyte pole located opposite
remarkable age-dependent changes in pinopsin immunoreactivity in the pineal organ of chickens; however, this study covered only 1- to 2month-old young chickens. Concerning pinopsin in adult birds, only one study was performed on pigeons (Okano et al., 1997). Our study is the first one to investigate changes in pinopsin immunoreactivity in the avian pineal gland during post-hatching development from hatching to maturity. The obtained data provide clear evidence for the reduction of pinopsin immunoreactivity in rudimentary photoreceptor pinealocytes after sexual maturation of turkeys. This process overlaps with the observation of ultrastructure regressive changes of these cells, including reduction of the apical extension (Petrusewicz-Kosińska et al., 2019). It also coincides with the accumulation of immunoreactive cellular debris in the follicle lumen. On the other hand, the number of pinopsin immunopositive string-shape or spherical structures, located in the outer part of the follicle wall in the turkey pineal organ, prominently increases with age. This process correlates with a large increase in the number of secretory pinealocytes during postnatal development of the turkey pineal gland. These 9
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Fig. 10. Secretion of melatonin (mean) from the pineal organs of 40-week-old turkeys incubated under a 12 L:12 D cycle with the photophase from 07.00 to 19.00 or a reversed cycle of 12 D:12 L during three consecutive days and then in continuous darkness for two days.
12-week-old geese and 9-month-old ducks to a reversed dark-light cycle takes more than two days (Ohshima and Matsuo, 1984; Prusik et al., 2015; Ziółkowska, 2015). A change in the light-dark cycle also affects the circadian oscillator in pinealocytes of adult turkeys.
to the nucleus. Our data demonstrates that the mechanism responsible for pinopsin distribution acts in a single cell and is independent of the localization of the cell in the follicle. The results obtained in our experiment showed that all pinealocytes in the turkey pineal organ are photosensitive and showed apparent polarization in vitro. Taking into account the morphology of parafollicular cells in the chicken, it was suggested that these cells are modified photoreceptor pinealocytes with a more simplified structure than that of rudimentary photoreceptor cells (Ohshima and Matsuo, 1984; Hirunagi et al., 1997). Our data provide strong and direct evidence for the photoreceptor nature of secretory pinealocytes and support the above-mentioned suggestion. The changes in organization of the turkey pineal organ during postembryonic development offer the unique possibility of performing physiological studies on the glands, in which melatonin-synthetizing cells are represented almost exclusively by well-developed rudimentary receptor pinealocytes (in 4 – 12-week-old birds) or by regressed rudimentary receptor pinealocytes and highly numerous secretory cells (in 40–56 weeks old birds) (Przybylska-Gornowicz et al., 2005; Petrusewicz-Kosińska et al., 2019). In the present study, we compared the role of direct photoreception in the regulation of melatonin secretion in the organs of 10-week-old and 40-week-old turkeys. The obtained results show that the pineal organs of young birds are much more sensitive to light exposition at night than the pineal organs of adult turkeys. Short light exposition during scotophase induced a decrease in melatonin secretion in the pineal organs of young turkeys but not in adult birds. These data demonstrate, for the first time, functional differences between the two kinds of pinopsin-containing cells: rudimentary receptor pinealocytes with an internal organization resembling retinal photoreceptors are more light-sensitive than secretory pinealocytes resembling neurons. It is worth noting that the pineal organs of adult turkeys secrete melatonin in a well-entrained diurnal rhythm, with slightly lower amplitude than the glands of turkeys 10–14 weeks of age (Prusik and Lewczuk, 2019). For comparison, entrainment to a light-dark cycle is much weaker in the pineal organs of young ducks (Prusik et al., 2015), and especially in young geese (Ziółkowska, 2015), than in adult turkeys. It should also be underlined that despite the lower light sensitivity, the pineal organs of adult turkeys adopt their secretory activity to the reversed light-dark cycle during the second day of culture in 12 D:12 L conditions, as do the pineal organs of young turkeys. Such adaptation in the pineal organ pinealocytes of 15-week-old chickens,
5. Conclusions Our results showed that pinopsin is present in the turkey pineal organ during the entire period of postnatal development, from hatching to maturity; however, its distribution changes during this time. Agedependent reduction of pinopsin immunoreactive apical extensions of rudimentary photoreceptor pinealocytes occurs together with a simultaneous increase in pinopsin-immunopositive elements in the outer part of the follicle wall, composed of secretory pinealocytes. All pinealocytes in the turkey pineal organ are photoreceptive; however, secretory pinealocytes are less light-sensitive than rudimentary receptor pinealocytes. The autonomous regulatory mechanisms, including direct photoreception and endogenous rhythm generation, work well in the pineal organs of adult turkeys. Funding This study was founded by KNOW (Leading National Research Centre) Scientific Consortium “Healthy Animal – Safe Food” decision of Ministry of Science And Higher Education no. 05-1/KNOW2/2015. The publication costs were supported by the Minister of Science and Higher Education under the program entitled "Regional Initiative of Excellence" for the years 2019–2022, Project No. 010/RID/2018/19; the amount of funding was 12.000.000 PLN. Declaration of Competing Interest The authors declare that they have no conflicts of interest. Acknowledgments The authors wish to express their deep thanks to Professor Yoshitaka Fukada and Professor Toshiyuki Okano from The University of Tokyo, Japan for the gift of pinopsin antiserum. The authors also would like to thank Dr Andrew Foldes from the Division of Animal Production, CSIRO, Blacktown, Australia for the gift of anti-melatonin antibodies. 10
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The authors wish to express their deep thanks to Dr Pierre Voisin from The University in Poitiers, France for the gift of ASMT antiserum.
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