Cell Tiss. Res. 184, 133-137 (1977)
Cell and Tissue Research 9 by Springer-Verlag 1977
Short Communication
An Unusual Organelle in the Pineal Gland of the Rat M. Evelyn McNeill* Department of Anatomy, School of Medicine, East Carolina University, Greenville, North Carolina, U.S.A.
Summary. An electron microscopic survey of pinealocytes from normal rats revealed a highly organized arrangement of cytoplasmic tubules. Such tubules had been previously observed in normal rats (Lin, 1967) and in rats after melatonin administration or two weeks exposure to darkness (Freire and Cardinali, 1975). In a later publication the presence of the tubules was attributed to experimental manipulation resulting in infertility (Gusek, 1976). The present study resolves the discrepancy in the literature by establishing that the tubular organelle does indeed occur in untreated male rats, but rather rarely. Key words: Pineal - Rat - Unusual organelle - Ultrastructure.
Introduction In an electron microscopic study, Lin (1967) observed for the first time a peculiar and highly structured configuration of tubules in the cytoplasm of rat pinealocytes. He referred to the structure as a "canaliculate lamellar body" and found it in pineals of both male and female rats between two months and two years of age including rats in late pregnancy, animals maintained either in continuous light or in constant darkness for one week, and in untreated normal rats. Similar structures ("annulate lamellae") were observed by Freire and Cardinali (1975, see Fig. 13) after melatonin administration or exposure to darkness for two weeks. The same configuration ("Mikrotubuli") was recently reported by Gusek (1976. see p. 144) in sexually mature male rats after hormonal castration by cyproterone acetate. He found them only in animals treated with cyproterone acetate. The Send offprint requests to: M. Evelyn McNeill, Ph.D., Department of Anatomy, School of Medicine,
East Carolina University, Greenville, North Carolina 27834, U.S.A. * The author expresses her sincere appreciation to Professor J. Ari~ns Kappers and Professor H.-S. Lin for their suggestions concerning this paper, and to Nicki Smith and Dan Whitehead for skilled technical assistance
134
M.E.McNeill
purpose of the present study was to investigate the pineal's ultrastructure in untreated male rats with special emphasis on the search for the canaliculate lamellar body described by Lin (1967).
Materials and Methods Twenty two normal adult male Holtzman rats, housed in individual cages under diurnal artificial lighting (lights on from 7:00A.M.-7:00P.M.) and provided water and commercial lab chow ad libitum, were decapitated between 10: 00 A.M. and noon during May, June and July. The pineals were fixed in 2.5 % cacodylate-buffered glutaraldehyde for two hours, and postfixed for one hour in 2 % phosphate buffered osmium tetroxide, stained en bloc with uranyl acetate-maleic acid, dehydrated, and oriented in Epon to be sectioned horizontally, frontally or sagittally. Thin sections were stained with lead citrate and examined with a Philips 201 electron microscope.
Results and Discussion
The canaliculate lamellar body was observed in four of the 22 glands surveyed. When present it lies consistently in close proximity to lipid droplets (Fig. 1) and to rough endoplasmic reticulum (Fig. 2). Cytoplasmic aggregates of ribosomes accompany the structure (Fig. 2). The characteristic and uniform appearance of the organelle is that of closely packed lamellae stacked at right angles to a membrane containing periodically spaced annular pores. The average diameter of the pores is 550 A, the distance between being about half the diameter of the pores. Intercellular spaces, canaliculi, and the canaliculate lamellar body are shown in relation to the perivascular space (Fig. 1). Canaliculi can be distinguished from perivascular spaces as they appear empty in electron micrographs (Fig. 1). The configuration of this organelle with precisely arranged tubules running in a plane perpendicular to the lamellae is so characteristic that it is unmistakenly the organelle described by Lin (1967), by Freire and Cardinali (1975), and by Gusek (1976). The occurrence of these tubules and a possible functional role are considerations which appear worthy of attention. While the organelle is seen in pinealocytes of untreated rats, it does not occur as consistently as other organelles. Neither Ari~ns Kappers' (1969_) nor Wolfe's (1965) extensive studies of the rat pineal depict the exact structure in question though the "cisternal grille" in Figure 20 of Wolfe's article is somewhat comparable as pointed out by Lin (1967). Lin (1967) stated that many cells were surveyed before the canaliculate lamellar body was noticed. He observed the structure in both normal and experimental rats but did not comment on its incidence. A plethora of references in the literature fails to describe the organelle in other animals. The pineals in Lin's study were obtained from male and female Long-Evans rats; those in Gusek's and in Freire and Cardinali's studies from Wistar males, and those in the present study from Holtzman males. The paucity of its reported incidence therefore is not due to strain or sex specificity. The functional state of the animal may also determine the incidence of the structure. It is conceivable that the season of the year, hour of sacrifice, and stage of sexual maturity might influence the occurrence of the organelle. Gusek (1976)
Fig. 1. Canaliculi at left seen as empty, widened intercellular spaces, in part surrounded by organelle in question. Note amorphous material in perivascular space (PVS) and lipid (L). x 24,500. Fig. 2. Canaliculate lamellar body. Also note free ribosomes (R) and granular endoplasmic reticulum (GER). x 71,250
136
M.E.McNeill
compared hormonally castrated rats with surgically castrated animals of the same strain. Hormonal castration was effected by the administration of cyproterone acetate and "mikrotubuli" were found only in cyproterone-acetate treated animals. While it is possible that either the incidence or the extent of the tubular arrangement is altered by castration, the presence of the structure, per se, is not dependent upon experimental castration as shown by the present study. Cytoplasmic annulate lamellae bearing some resemblance to the organelle under consideration have been observed in an in vitro cell strain established from a human epidermoid carcinoma (Kumegawa et al., 1967); yet annulate lamellae described in hamster pinealocytes (Bucana et al., 1971; Lin et al., 1975) bear no resemblance to the structure in question. Annulate lamellae occasionally observed by Karasek et al. (1976) in orchidectomized rats show a periodicity of cisternae comparable to that shown in the present study. The organelle's exquisitely regular tubular arrangement, as well as its position near lipid and endoplasmic reticulum, are highly suggestive of a metabolic function. In the rat the lipid content of the pineal fluctuates with the estrous cycle (Zweens, 1963); therefore, it is not improbable that the incidence or extent of this peculiar configuration of tubules may also fluctuate. The organelle might be an elaborate modification of the endoplasmic reticulum or a sub-form of annulate lamellae. Since it has such a distinct profile, however, it may prove to be an organelle in its own right. In summary, a unique and elusive organelle command~ attention in a gland whose secretory mechanism also remains enigmatic. Morphological studies have offered very little information regarding the mode of secretion of either melatonin or of polypeptides. In fact, whether the pineal delivers it products into the blood vascular system or utilizes the cerebrospinal fluid and ventricular cavities for transport continues to be open to question. In Ari6ns Kappers' (1969) discussion of the mammalian pineal, he argues strongly for the blood vascular route while Pavel (1971) describes a direct delivery of arginine vasotocin into the cerebrospinal fluid. The proximity of the organelle to lipid, granular endoplasmic reticulum and to pineal canaliculi, and the current interest in pineal polypeptides provide a stimulus for investigating the organelle's presumptive involvement in secretory function. Cytochemical studies are necessary to further characterize it. The present study confirms the organelle's occurrence in male rats unrelated to experimental manipulation.
References Ari~ns Kappers, J.: The mammalian pineal organ. J. Neuro-Visc. Relat., Suppl. IX, 140-184 (1969) Bucana, C.D., Nadakavukaren, M.J., Frehn, J.L.: Annulate lamellae in hamster pineal gland. Tissue & Cell 3, 405-412 (1971) Freire, F., Cardinali, D.F.: Effects of melatonin treatment and environmental lighting on the ultrastructural appearance, melatonin synthesis, norepinephrine turnover and microtubule protein content of the rat pineal gland. J, Neural Transmiss. 37, 237-257 (1975) Gusek, W.: Die Feinstruktur der Rattenzirbel und ihr Verhalten unter EinfluB von Antiandrogen und nach Kastration. Endokrinologie 67, 129-151 (1976) Karasek, M., Pawlikowski, M., Ari6ns Kappers, J., Stepien, H.: Influence of castration followed by
Unusual Pineal Organelle
137
administration of LH-RH on the ultrastructure of rat pinealocytes. Cell Tiss,Res. 167, 325-339 (1976) Kumegawa, M., Cattoni, M., Rose, G.G.: Electron microscopy of oral cells in vitro. J. Cell Biol. 34, 897-901 (1967) Lin, H.-S.: Peculiar configuration of agranular reticulum (canaliculate lamellar body) in the rat pinealocyte. J. Cell Biol. 33, 15-25 (1967) Lin, H.-S., Hwang, B.-H., Tseng, C.-Y.: Fine structural changes in the hamster pineal gland after blinding and superior cervical ganglionectomy. Cell Tiss. Res.158, 285-299 (1975) Pavel, S.: Evidence for the ependymal origin of arginine vasotocin in the bovine pineal gland. Endocrinology 89, 613-614 (1971) Wolfe, D.E.: The epiphyseal cell: an electron-microscopic study of its intercellular relationships and intracellular morphology in the pineal body of the albino rat. Progr. Brain Res. 10, 332-386 (1965) Zweens, J.: Influence of the estrous cycle and ovariectomy on the phospholipid content of the pineal gland. Nature (Lond.) 197, 1114 1115 (1963) Accepted June 20, 1977
Cell and Tissue Research 9 by Springer-Verlag 1977
Short Communication
An Unusual Organelle in the Pineal Gland of the Rat M. Evelyn McNeill* Department of Anatomy, School of Medicine, East Carolina University, Greenville, North Carolina, U.S.A.
Summary. An electron microscopic survey of pinealocytes from normal rats revealed a highly organized arrangement of cytoplasmic tubules. Such tubules had been previously observed in normal rats (Lin, 1967) and in rats after melatonin administration or two weeks exposure to darkness (Freire and Cardinali, 1975). In a later publication the presence of the tubules was attributed to experimental manipulation resulting in infertility (Gusek, 1976). The present study resolves the discrepancy in the literature by establishing that the tubular organelle does indeed occur in untreated male rats, but rather rarely. Key words: Pineal - Rat - Unusual organelle - Ultrastructure.
Introduction In an electron microscopic study, Lin (1967) observed for the first time a peculiar and highly structured configuration of tubules in the cytoplasm of rat pinealocytes. He referred to the structure as a "canaliculate lamellar body" and found it in pineals of both male and female rats between two months and two years of age including rats in late pregnancy, animals maintained either in continuous light or in constant darkness for one week, and in untreated normal rats. Similar structures ("annulate lamellae") were observed by Freire and Cardinali (1975, see Fig. 13) after melatonin administration or exposure to darkness for two weeks. The same configuration ("Mikrotubuli") was recently reported by Gusek (1976. see p. 144) in sexually mature male rats after hormonal castration by cyproterone acetate. He found them only in animals treated with cyproterone acetate. The Send offprint requests to: M. Evelyn McNeill, Ph.D., Department of Anatomy, School of Medicine,
East Carolina University, Greenville, North Carolina 27834, U.S.A. * The author expresses her sincere appreciation to Professor J. Ari~ns Kappers and Professor H.-S. Lin for their suggestions concerning this paper, and to Nicki Smith and Dan Whitehead for skilled technical assistance
134
M.E.McNeill
purpose of the present study was to investigate the pineal's ultrastructure in untreated male rats with special emphasis on the search for the canaliculate lamellar body described by Lin (1967).
Materials and Methods Twenty two normal adult male Holtzman rats, housed in individual cages under diurnal artificial lighting (lights on from 7:00A.M.-7:00P.M.) and provided water and commercial lab chow ad libitum, were decapitated between 10: 00 A.M. and noon during May, June and July. The pineals were fixed in 2.5 % cacodylate-buffered glutaraldehyde for two hours, and postfixed for one hour in 2 % phosphate buffered osmium tetroxide, stained en bloc with uranyl acetate-maleic acid, dehydrated, and oriented in Epon to be sectioned horizontally, frontally or sagittally. Thin sections were stained with lead citrate and examined with a Philips 201 electron microscope.
Results and Discussion
The canaliculate lamellar body was observed in four of the 22 glands surveyed. When present it lies consistently in close proximity to lipid droplets (Fig. 1) and to rough endoplasmic reticulum (Fig. 2). Cytoplasmic aggregates of ribosomes accompany the structure (Fig. 2). The characteristic and uniform appearance of the organelle is that of closely packed lamellae stacked at right angles to a membrane containing periodically spaced annular pores. The average diameter of the pores is 550 A, the distance between being about half the diameter of the pores. Intercellular spaces, canaliculi, and the canaliculate lamellar body are shown in relation to the perivascular space (Fig. 1). Canaliculi can be distinguished from perivascular spaces as they appear empty in electron micrographs (Fig. 1). The configuration of this organelle with precisely arranged tubules running in a plane perpendicular to the lamellae is so characteristic that it is unmistakenly the organelle described by Lin (1967), by Freire and Cardinali (1975), and by Gusek (1976). The occurrence of these tubules and a possible functional role are considerations which appear worthy of attention. While the organelle is seen in pinealocytes of untreated rats, it does not occur as consistently as other organelles. Neither Ari~ns Kappers' (1969_) nor Wolfe's (1965) extensive studies of the rat pineal depict the exact structure in question though the "cisternal grille" in Figure 20 of Wolfe's article is somewhat comparable as pointed out by Lin (1967). Lin (1967) stated that many cells were surveyed before the canaliculate lamellar body was noticed. He observed the structure in both normal and experimental rats but did not comment on its incidence. A plethora of references in the literature fails to describe the organelle in other animals. The pineals in Lin's study were obtained from male and female Long-Evans rats; those in Gusek's and in Freire and Cardinali's studies from Wistar males, and those in the present study from Holtzman males. The paucity of its reported incidence therefore is not due to strain or sex specificity. The functional state of the animal may also determine the incidence of the structure. It is conceivable that the season of the year, hour of sacrifice, and stage of sexual maturity might influence the occurrence of the organelle. Gusek (1976)
Fig. 1. Canaliculi at left seen as empty, widened intercellular spaces, in part surrounded by organelle in question. Note amorphous material in perivascular space (PVS) and lipid (L). x 24,500. Fig. 2. Canaliculate lamellar body. Also note free ribosomes (R) and granular endoplasmic reticulum (GER). x 71,250
136
M.E.McNeill
compared hormonally castrated rats with surgically castrated animals of the same strain. Hormonal castration was effected by the administration of cyproterone acetate and "mikrotubuli" were found only in cyproterone-acetate treated animals. While it is possible that either the incidence or the extent of the tubular arrangement is altered by castration, the presence of the structure, per se, is not dependent upon experimental castration as shown by the present study. Cytoplasmic annulate lamellae bearing some resemblance to the organelle under consideration have been observed in an in vitro cell strain established from a human epidermoid carcinoma (Kumegawa et al., 1967); yet annulate lamellae described in hamster pinealocytes (Bucana et al., 1971; Lin et al., 1975) bear no resemblance to the structure in question. Annulate lamellae occasionally observed by Karasek et al. (1976) in orchidectomized rats show a periodicity of cisternae comparable to that shown in the present study. The organelle's exquisitely regular tubular arrangement, as well as its position near lipid and endoplasmic reticulum, are highly suggestive of a metabolic function. In the rat the lipid content of the pineal fluctuates with the estrous cycle (Zweens, 1963); therefore, it is not improbable that the incidence or extent of this peculiar configuration of tubules may also fluctuate. The organelle might be an elaborate modification of the endoplasmic reticulum or a sub-form of annulate lamellae. Since it has such a distinct profile, however, it may prove to be an organelle in its own right. In summary, a unique and elusive organelle command~ attention in a gland whose secretory mechanism also remains enigmatic. Morphological studies have offered very little information regarding the mode of secretion of either melatonin or of polypeptides. In fact, whether the pineal delivers it products into the blood vascular system or utilizes the cerebrospinal fluid and ventricular cavities for transport continues to be open to question. In Ari6ns Kappers' (1969) discussion of the mammalian pineal, he argues strongly for the blood vascular route while Pavel (1971) describes a direct delivery of arginine vasotocin into the cerebrospinal fluid. The proximity of the organelle to lipid, granular endoplasmic reticulum and to pineal canaliculi, and the current interest in pineal polypeptides provide a stimulus for investigating the organelle's presumptive involvement in secretory function. Cytochemical studies are necessary to further characterize it. The present study confirms the organelle's occurrence in male rats unrelated to experimental manipulation.
References Ari~ns Kappers, J.: The mammalian pineal organ. J. Neuro-Visc. Relat., Suppl. IX, 140-184 (1969) Bucana, C.D., Nadakavukaren, M.J., Frehn, J.L.: Annulate lamellae in hamster pineal gland. Tissue & Cell 3, 405-412 (1971) Freire, F., Cardinali, D.F.: Effects of melatonin treatment and environmental lighting on the ultrastructural appearance, melatonin synthesis, norepinephrine turnover and microtubule protein content of the rat pineal gland. J, Neural Transmiss. 37, 237-257 (1975) Gusek, W.: Die Feinstruktur der Rattenzirbel und ihr Verhalten unter EinfluB von Antiandrogen und nach Kastration. Endokrinologie 67, 129-151 (1976) Karasek, M., Pawlikowski, M., Ari6ns Kappers, J., Stepien, H.: Influence of castration followed by
Unusual Pineal Organelle
137
administration of LH-RH on the ultrastructure of rat pinealocytes. Cell Tiss,Res. 167, 325-339 (1976) Kumegawa, M., Cattoni, M., Rose, G.G.: Electron microscopy of oral cells in vitro. J. Cell Biol. 34, 897-901 (1967) Lin, H.-S.: Peculiar configuration of agranular reticulum (canaliculate lamellar body) in the rat pinealocyte. J. Cell Biol. 33, 15-25 (1967) Lin, H.-S., Hwang, B.-H., Tseng, C.-Y.: Fine structural changes in the hamster pineal gland after blinding and superior cervical ganglionectomy. Cell Tiss. Res.158, 285-299 (1975) Pavel, S.: Evidence for the ependymal origin of arginine vasotocin in the bovine pineal gland. Endocrinology 89, 613-614 (1971) Wolfe, D.E.: The epiphyseal cell: an electron-microscopic study of its intercellular relationships and intracellular morphology in the pineal body of the albino rat. Progr. Brain Res. 10, 332-386 (1965) Zweens, J.: Influence of the estrous cycle and ovariectomy on the phospholipid content of the pineal gland. Nature (Lond.) 197, 1114 1115 (1963) Accepted June 20, 1977
