Jou~ of. N~ral Tran,m~sfim. (~ by Springer-Verlag 1977. The Pineal Gland of Nocturnal Mammals. I. The Pinealocytes of the Bat (Nyctalus noctula, Schreber).
J o u ~ of
J. Neural Transmission 40, 47--68 (1977)
N ~ r a l Tran,m~sfim (~ by Springer-Verlag 1977
The Pineal Gland of Nocturnal M a m m a l s I. T h e Pinealocytes of the B a t ( N y c t a l u s
n o c t u l a , Schreber)
P. Pevet, J. Ari~ns Kappers, and A. M. Vofite* The Netherlands Central Institute for Brain Research, Amsterdam, The Netherlands, and ':'Institute for Animal Ecology and Taxonomy, University of Utrecht, The Netherlands With 30 Figures Received September 16, 1976
Summary The ultrastructure of the pinealocytes of noctule bats, mammals which live most of the time in darkness or very low light intensities, was examined and compared with the pinealocytes of other mammals. Two different populations of pinealocytes (I and II) were observed. They differ in general aspect, in location and especially in their content of cell organelles involved in synthetic processes. Mitochondria, ribosomes, lysosomes and lipid inclusions were present in the perikaryon of pinealocytes of both populations. In the pinealocytes of population I some granular vesicles, of presumed Golgi origin, and some other structures were observed. Pinealocytes of population II are characterized by many glycogen granules, more or less associated with a large vacuolar system. Moreover, some small vacuoles originating from cisterns of the granular endoplasmic reticulum and containing flocculent material of a moderate electron density are described. The possibility is discussed that these small vacuoles are involved in one of the secretory processes of the pineal gland while the granular vesicles of the pinealocyte of the population I are the products of another. Key words: Pinealocytes, bat, ultrastructure, secretion.
Introduction One of the important physiological effects of the pineal is to synchronize the reproductive activity of seasonal breeding species with the proper season of the year (Reiter, 1973 a, b, 1974 a, b, c, 1975; Reiter et al., 1975; Pevet, 1976). It thus seems important to
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P. Pevet, J. Ari~ns Kappers, and A. M.Vofite:
study the pineal gland of mammals living under natural conditions, because, given the fact that the pineal suppresses gonadal function and that darkness enhances pineal activity (see Kappers, 1969; Reiter, i974 b; Reiter et aI., t975, for references), it can be expected that their pineal gland is highly functionally active, specially during the winter months when daylength is shorter. At the ultrastructural level, few studies of t!le pineal gland have dealt with species living in natural conditions such as the antartic seal (Cuello, 1973), dormouse (Roux et al., 1974), ground squirrel (Matsushima and Reiter, 1975; Povlishock et al., 1975), hedgehog (Pevet, 1972, 1975, 1976; Pevet and Saboureau, 1973, 1974), mole (Pevet, 1974, 1975; Pevet and Smith, 1975; Pevet et al., I976 a), mole-rat (Miline et al., 1975 a, b; Pevet, 1976; Pevet et aL, 1976 b) and pocket gopher (Sheridan and Reiter, i973). Since these animals are active at night and sleep in darkness during the day (Stuiter and Van Heerdt, 1966), it would seem that also an investigation of the bat pineal would be rewarding in this respect. The present report describes the fine structure of the pinealocytes of one bat species, the noctule bat (Nyctalus noctuta) of which the sexual cycle is well-known (Racey, 1974). Materials and Methods The two female noctule bats used in this study were caught in the Netherlands. One was removed from its hibernaculum in a tree which had been felled on February 5th, 1975. After capture this animal was taken to the laboratory, where it was fed with larvae of Tenebrio molitor (mealworms) and was kept in natural lighting until March 7th. The other bat was kept in captivity in the laboratory from September 22nd, 1973, until March 7th, 1975. It was used several times for hibernating and activity experiments in total darkness or artificial lightdark rhythms. During the last months of its life this bat was submitted to natural lighting. This animal was also fed with living meal-worms, together with a mixture of egg-yolk, minced meal-worms, milk powder, vitamins, minerals and trace elements. The bats were decapitated under ether anaesthesia. The skuli was opened and the region of the pineal flooded with cold fxative (2.5 ~ glutaraldehyde in 0.1 M phosphate buffer, pH 7.25, at 4 ~ The time elapsed between decapitation and flooding of the gland was less than one minute. After its removal, the gland was placed in the same fixative at a temperature of 4 ~ for a 1/2 hour. The organs were then postfixed in I ~ OsO4 and 0.2 M phosphate buffer (pH 7.25) at room temperature during one hour. The glands were dehydrated and embedded in aratdite (Glauert and Glauert, 1958).
Noctule Bat Pinealocytes
49
Thin sections were cut on a LKB ultramicrotome with glass knives. They were stained with uranyl acetate and lead citrate (Reynolds, 1963; Venable and Coggeshall, 1965). On some thin sections, the periodic acid-thiocarbohydrazide-silver proteinate (PATAg) technique of Thiery (1967) was applied to detect glycogen granules. Observations were carried out on a Philips 200 electron microscope. Results
The parenchyma of the pineal gland primarily consists of pinealocytes with a few dispersed glial cells. Two different populations of pinealocytes, differing in location, in the appearance of their nuclei and especially in their secretory activity were observed.
Pinealocytes of Population I These pinealocytes are irregular in shape, showing cytoplasmic processes emerging from their cell bodies. They are homogeneously dispersed in the pineal tissue and represent 70--80 % of the total amount of pinealocytes. The nucleus is large, oval or polygonal in shape (Figs. 1, 13), but sometimes it is lobutated (Fig. 6). Chromatin is widely dispersed in the nuclear matrix, but numerous chromatin aggregations are seen, especially in a zone adjoining the nuclear envelope (Figs. 1, 13). Many Goigi apparatuses are usually widely dispersed around the nucleus. Each Golgi apparatus consists of a system of flattened sacs associated with a population of vesicles of varying diameter (Fig. 3). Some of these vesicles are granular measuring (60--200 nm) in diameter (Figs. 3, 4). Granular vesicles are also observed throughout the cytoplasm and the processes of the cells. Numerous elongated or spherical mitochondria are found within the cytoplasm (Figs. 10, 13). Membrane-bound dense bodies, presumably lysosomaI structures, possess a single membrane. A granular matrix and rare lipid droplets are also observed. Smooth endoplasmic reticulum is rare. If, however, present, it demonstrates an unusual appearance consisting of very flat cisterns (Figs. 5, 6). These structures most likely originate from the granular endoplasmic reticulum (Figs. 8, 9). In some cells peculiar organized structures are observed either lying freely in the cytoplasm (Fig. 12) or in close connection with the special smooth endoplasmic reticulum earlier described (Figs. 1, 7). Numerous single ribosomes, polysomes and cisterns of the granular endoplasmic reticuium are found scattered throughout the .lournai o~ Neural Transmission 40/1
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P. Pevet, J. Ari~ns Kappers, and A. M. Vo6te:
cytoplasm (Fig. 13). Often, condensation of material is observed between two cisterns (Figs. 9--11, 13). Subsurface cisterns (Fig. 2) are situated close to the plasmaiemma in the perikaryon and also within pinealocyte processes. Sometimes, two subsurface cisterns present in adjacent pineatocytes face each other (Fig. 2). Ciliated structures are found in numerous pinealocytes (Fig. 13). Very often they present a characteristic aspect (Fig. 14) and when seen in cross section they exhibit a 9 + 0 arrangement of paired tubules (Fig. 15). Centrioles (Fig. 16) are also present either in association with ciliated structures or not. Like in other mammals, the processes of pinealocytes of population I are very long. They contain many microtubules, running parallel to the long axis of the process, and some organelles, particularly mitochondria.
Pinealocytes of Population II The general appearance of the pinealocytes of population It is similar to that os population I. Although they are found throughout the pineal gland most are located close to a perivascular space (Figs. 1, 24). Like in the pinealocytes of population I, the nucleus is large, oval or polygonal in shape. The chromatin, however, is finely dispersed in the nuclear matrix not showing aggregations (Figs. 1, 2t). Many organelles present in the cytoplasm such as mitochondria, lysosomal structures, Golgi apparatus and its associated vesicles, lipid droplets, subsurface cisterns and ciliary derivatives with their 9 + 0 arrangement of paired tubules are identical to those described in the pinealocytes of population I. The ciliary derivatives are, however, fewer in number and more lipid droplets are present than in the pinealocytes of population I. Moreover, the peculiar shape of the smooth endoplasmic reticulum (Fig. 5), the peculiar organized struc~ tures (Fig. 12) and the granular vesicles which have been described in other pinealocytes are never observed in those of population II. These pinealocytes are essentially characterized by many opaque cytoplasmic granules which are larger than ribosomes (Figs. t, 19 to 22). In some cells enormous numbers of these granules occur (Fig. 1). Their intense staining with PATAg (Thiery, 1967) (Figs. 17, 18) showed that these granules consist of glycogen. The formation of numerous vacuoles has also been observed in the cytoplasm (Figs. 1, 21, 22). It is evident that they grow larger by fusion with each other (Fig. 19) and that glycogen granules are involved, at least in part, in their formation or maturation (Figs. 1, 19, 22). It seems that these vacuoles release their content into the extracellular space (Fig. 20).
Noctule Bat Pinealocytes
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The granular endoplasmic reticulum of these ceils also plays an important rote. Often it forms complexes in the vicinity of the nucleus which are composed of some parallel cisterns of granular reticutum and numerous small vacuoles situated between these cisterns (Figs. 21, 22). These small vacuoles contain flocculent material of moderate electron density (Figs. 22, 23). Some micrographs suggest that these vacuoles originate from the cisterns (Figs. 22, 23). The cytoplasm of some pinealocytes of population II contains numerous pigment granules (Fig. 24). Cisterns of the granular endoplasmic reticulum, concentrically arranged, are also observed in numerous pineatoeyte processes (Fig. 25). In rare cases some small vacuoles containing flocculent material of moderate electron density are present between them (Fig. 26). In other cases these concentric cisterns are in close connection with the peculiar shaped smooth endoplasmic reticulum (Fig. 27) or with the peculiar organized structures (Fig. 12) earlier described (Fig. 28). It has not been possible to determine whether these processes belong to either pinealocytes of type I or type II (see Discussion). Concentric lamellae (Fig. 29), concentrated in special areas of the pineal parenchyma (Fig. 30), are also very rarely observed. It is not known whether these structures are present in either the processes of pinealocytes of population I, or in those of population II, or in both. Table 1 summarizes the findings. Table 1. Characteristics o] the two different populations of pinealocytes Population I location
nucleus
perikaryon
-- homogeneously dispersed in the paren&yma -
-
-
-- practically always located close to a perivascular space
chromatin aggregations close to the nuclear envelope
granular vesicles -- smooth endoplasmic reticulum of a peculiar shape -
Population II
&romatin finely dispersed
--
e n o r m o u s
an2ount o f
glycogen granules vacuolar system -- small vacuoles originated from granular endoplasmic reticutum and containing a flocculent and moderate etectron dense material 4*
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P. Pevet, J. Ari~ns Kappers, and A. M.Vofite:
Noctule Bat Pinealocytes
53
Discussion It is now generally accepted that the mammalian pineal gland is evidently an endocrine gland, although neither its active principles nor the target organs of these principles are exactly known. Signs of secretory activity have been sought for in the pineal of the noctule bat. Until now, in only four mammalian species, the rabbit (Rornijn, 1972), the mouse (Upson et al., 1976), the mole-rat (Pevet et al., 1976 b) and the noctule bat, two different populations of pinealocytes have been described. In view of the endocrine function of the pineal gland, these observations raise problems, firstly because m a n y authors have used the terms "light" and "dark" pinealocytes to describe two different categories of pinealocytes and, secondly, because in many mammalian species only one population of pineatocytes has been observed. These problems are extensively discussed elsewhere (Pevet, 1977). In the present paper we will restrict ourselves to the following. Numerous pinealocytes of population I and some of population II possess ciliary derivatives characterized by a 9 + 0 tubular pattern. Moreover, some rare pinealocytes, especially in population I, were seen showing a polarity of the cellular body, this polarity being similar to that described by Collin (1969--1971) in secretory rudimentary photoreceptor cells of non-mammalian vertebrate pineal glands. Also, in some rare cases outer segments were observed. Thus, like in mole (Pevet and Collin, 1976), different types of pinealocytes are present in both populations. These results will be exposed elsewhere (in preparation). Clear vesicIes were observed in association with the Golgi apparatus in the cytoplasm of the ceil body in both pinealocyte Fig. t. Part of two pineatocytes, one of population I (PI) and the other of II (PIt). The nucleus (n) of the pinealocyte of population I shows a dispersed pattern of chromatin in its marginal zone. In the cytoplasm, peculiar structures (small arrow) can be distinguished. Near the pinealocyte of population II a capillary (CAP) is present and in its cytoplasm, aggregations of glycogen granules (big arrow) and vacuoles (*) are seen. 5< 15,300 Fig. 2. Subsurface cistern complex which consists of two cisterns (*) present in two pinealocytes and facing each other. Some ribosomes (small arrow) are situated on their membranes facing the interior of the cell. Between the two cisterns the plasma membranes of the two cells are observed (big arrows). X48,000 Fig. 3. Golgi apparatus (g). Many clear vesicles (v) are associated with the Golgi saccules. A granular vesicle (arrow) is observed near the Golgi complex. X 31,300 Fig. 4. Granular vesicle. Between the dense matrix and the membrane there is a clear halo (arrow). • 92,000
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P. Pevet, J. Ari~ns Kappers, and A. M. Vofite:
Noctule Bat Pinealocytes
55
populations. They have also been found in pinealocytes of several other mammals (see list in Pevet et al., 1976 b). However, the functional role and the biochemical nature of their content are not known. Granular vesicles, which were observed in the pinealocytes of all other mammalian species investigated and also in all principal epiphyseal cells of lower vertebrates (see list in Pevet et aI., 1976 b) have also been found in the pinealocytes of the noctule bat, but, until now, exclusively in those of population I. As some granular vesicles were observed in the vicinity of the Golgi apparatus, it is very probable that in the bat, like in other mammals, these structures are produced by the Golgi saccules. The granular vesicles apparently represent a secretory activity of the pineal gland (see Pevet, 1977). The peculiar packets of smooth endoplasmic reticulum found in the pinealocytes of population I (Figs. 5, 6) have only been described in two other species, the mole (Pevet, 1974, 1976) and the mole-rat (Pevet, 1976; Pevet et aI., 1976 b). Their function is unknown, but it is interesting to note that they were observed in 3 mammals in which the ultrastructural studies demonstrated an intensive synthetic activity, specially evident at the level of the granular endoplasmic reticulum. As, like in the mole-rat (Pevet et al., 1976 b), these elements are in close connection with cisterns of the granular endoplasmic reticulum, it is evident that they are involved in pinealocyte activity.
Fig. 5. Smooth endoplasmic reticulum showing a peculiar shape (*). It consists of a system of long and flattened cisterns. This system lies freely in the cytoplasm. X 32,000 Fig. 6. Pinealocyte of population I (P) characterized by the presence of smooth endoplasmic reticulum of a peculiar structure (-~). This picture is exceptional regarding the size of this structure, n = nucleus. 5
