Cell and Tissue Research

Cell Tissue Res (1981) 216:253-271

9 Springer-Verlag 1981

The Pineal Gland of Nocturnal Mammals II. The Ultrastructure of the Pineal Gland in the Pipistrelle Bat (Pipistrellus pipistrellus L.): Presence of Two Populations of Pinealocytes P. P6vet t'2 and P.A. Racey 3 1 The Netherlands Institute for Brain Research, Amsterdam, The Netherlands; 2 Department of Anatomy and Embryology,Universityof Amsterdam, Amsterdam, The Netherlands; 3 Department of Zoology, University of Aberdeen, Aberdeen, Scotland

Summary. In the pineal gland of the pipistrelle bat two different populations of pinealocytes and glial cells were observed electron microscopically. The pinealocytes of populations I and II differ in their content of metabolically active cell organelles. In the pinealocytes of population I, granular vesicles originating from the Golgi apparatus were found in the perikaryon and especially in the endings of the pinealocyte processes. Granular vesicles appeared to be more numerous in hibernating nulliparous females. The pinealocytes of population II are characterized by the presence of small cytoplasmic vacuoles, probably originating from cisternae o f the granular endoplasmic reticulum and containing flocculent material of moderate electron density. The classification of the pinealocytes belonging to population II is discussed. Key words: Pinealocytes - Cell populations - Bat - Ultrastructure

It has become increasingly evident that the pineal gland is principally involved in the long-term adaptation of reproductive function to environmental conditions, especially in seasonally breeding m a m m a l s (Reiter 1973, 1975, 1978; P6vet 1976, 1979; Vivien-Roels 1980). Since the quantity of light directly determines the functional state of the pineal and darkness enhances pineal activity (Kappers 1976), our recent research has been concentrated on fossorial and nocturnal m a m m a l s which, under natural conditions, are exposed to very little light. Send offprint requests to: Dr. P. P6vet, The Netherlands Institute for Brain Research, IJdijk 28, 1095 KJ Amsterdam, The Netherlands Acknowledgements. The authors wish to thank Mr. P.S. Wolters for his skillful technical assistance, and Miss J. Sels for secretarial aid. This collaboration was initiated with the aid of an SRC European short visit grant to P.A.R. The study was supported by the Foundation for Medical Research, the Netherlands (FUNGO, 13-35-33)

0302-766X/81/0216/0253/$03.80

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A m o n g n o c t u r n a l m a m m a l s , the bats o f temperate latitudes are of special interest. They roost in darkness d u r i n g the day a n d are active at night except d u r i n g winter a n d they are also seasonal breeders with a sexual cycle i n t e r r u p t e d by h i b e r n a t i o n (Racey 1979). Surprisingly, however, there have been few studies of bat pineal glands (Bozza 1927; Creutzfeldt 1912; K r a b b e 1920; L u n a 1921 ; Q u a y 1970, 1976; P6vet et al. 1977a, b; S t a m m e r 1972). The present report describes the fine structure of the pineal cells in the pipistrelle bat (Pipistrellus pipistrellusL.), the sexual cycle of which has been investigated in detail (Racey 1969, 1974; Racey a n d T a m 1974). I n the only other species of b a t studied electron microscopically, the noctule (P6vet et al. 1977a, b). two different p o p u l a t i o n s of pinealocytes were f o u n d ; thus, emphasis has been placed in the present study on the characterization of these cells in the pipistrelle.

Materials and Methods In the present study, 42 pipistrelle bats were used. They were captured in Great Britain between June 1976 and June 1977 (see Table 1). Animals were taken to the laboratory and killed within 4 days or maintained in an artificial hibernaculum for up to 2 months (Table 1). After decapitation the skull was opened and the region of the pineal was flooded with cold fixative (2.5 ~ glutaraldehyde in 0.1 M phosphate buffer, pH 7.25, 4 ~

Table 1. Animals used in the present investigation Reproductive condition

Place of capture

Date of capture

Date of death

Conditions between capture and death

Condition of bats at death

i0 pregnant 99 4 post-partum 99

Newtonmore Highland region

18 June 1976

21 June 1976

room temp.

active

3 parous 99 1 nulliparous 99 3 juvenile dd

Hatton, Grampian

20 August 1976

24 August 1976

room temp.

active

3 immature dd 1 nulliparous 9

Suffolk

3 March 1977

9 March 1977

5~

torpid

3 parous 99 3 adult dd

Suffolk

3 March 1977

6 April 1977

5~

torpid

4 nulliparous 99 3 adult dd

Suffolk

3 March 1977

12 May 1977

5~

torpid

5 pregnant 99

Inverurie Grampian

8 June 1977

9 June 1977

room temp.

active

Explanation: In June and August, bats were caught from their nursery roosts in the roof spaces of houses and maintained according to Racey (1970) until killed. In March, bats were caught from natural hibernacula and maintained in an artificial hibernaculum in the laboratory until they were killed. Bats naturally arise from hibernation during April, thus hibernation was considered to be artificially prolonged in those animals killed on 12 May 1977.

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255

Fig. 1. Pineal parenchyma. Light and dark pinealocytes of population I (P1): pinealocytes of population

II (PII). •

After removal, the gland was placed in the same fixative at a temperature of 4 ~C for 0.5 h. The organs were postfixed in 1 ~ OsO 4 in 0.2M phosphate buffer (pH 7.25) at room temperature for l h . The glands were then dehydrated and embedded in Araldite (Glauert and Glauert 1958). Thin sections were cut with glass knives and stained with uranyl acetate and lead citrate (Reynolds 1963; Venable and Coggeshall 1965). Observations were carried out on a Philips 200 electron microscope.

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Fig. 2. Pineal parenchymashowingseveralpinealocytesof population I. "Light" (LPI)and"dark" (DPI) pinealocytes of population I can be distinguished. Note the presence in the "dark" pinealocyte of a mitochondrion containing microfilaments(mit), a structure characteristic of pipistrelle pinealocytes of population I; n nucleus. • 25,000

Results The parenchyma of the pineal gland of the pipistrelle bat consists primarily of pinealocytes with very few dispersed glial cells. Two populations of pinealocytes differing especially in their secretory activity were observed.

Pinealocytes of Population I These pinealocytes are irregular in shape (Fig. 1), showing cytoplasmic processes emerging from their perikarya. They are homogeneously dispersed in the pineal tissue, account for 80 to 90 % of the total number of pinealocytes, and appear as both light and dark cells (Figs. 1,2). Because such cytological features as granular vesicles (GV) and mitochondria containing microfilaments (Fig. 2) are present in

Fig. 3. General view of pinealocytes of population II (Pll); g Golgi apparatus; mit mitochondria, n nucleus; nu nucleolus, x 14,000 Fig. 4. High magnification of the perikaryon of a pinealocyte of population II presented in Fig. 3. Note numerous vacuoles containing flocculent material of moderate electron density (arrows), the characteristic element of the pinealocytes of population II; lip lipid droplet; mit mitochondria, x 30,000

Fig. 5. Glial cell (GC). Note numerous glycogen granules (gg) and microfilaments (j') in the cell soma; n nucleus, x 13,000 Fig. 6. High magnification of the perikaryon of the gtial cell shown in Fig. 5. Note the bundle of parallelrunning filaments (/) and numerous glycogen granules (gg); g Golgi apparatus; mit mitochondria; n nucleus, x 30,000

Fig. 7. Processes o f pinealocytes of population I (P). Note granular vesicles (arrows). Between the granular vesicles numerous small clear vesicles (v) are also present, x 38,000 Fig. 8. Concentric lamellae (*) observed rarely in the pipistrelle pineal, x 15,600 Fig. 9. Granular vesicles (gv) located in the vicinity of the Golgi apparatus (g). Note the presence o f microfilaments (arrow) in one of the mitochondria (mit). x 27,300

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Fig. 10. Giant mitochondrion (mit) in a pinealocyte of population I characterized by the presence of a bundle of microfilaments (arrow). x 21,600

Fig. 11. Giant mitochondrion (mit) completely filled with parallel bundles of microfilaments (arrows). • 21,000 Figs. 12,13. Small mitochondria containing microfilaments

(arrows). x 28,000; x 20,000

Fig. 14. Presence of bundles of microfilaments in mitochondria, two of which are completely filled

(arrows). A large mitochondrion is composed of two parts, one containing parallel bundles of microfilaments (,). Normal mitochondrion (double arrow), x 45,000 Fig. 15. Two mitochondria containing microfilaments n nucleus. • 21,200

(arrows) apparently in a process of fusion;

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261

Fig. 16. Vacuole apparently associated with glycogen granules (asterisk) in a pinealocyte of population I (P/); gg glycogen granules, x 22,800 Fig. 17. Vacuole apparently associated with glycogen granules (asterisk) in a pinealocyte of population II (Pit). Note vacuoles containing flocculent material (arrows), the characteristic element ofpinealocytes of population II; gg glycogen granules; mit mitochondria; n nucleus, x 30,000 both these cell types and because stages intermediate in electron density are also present, we consider the light and d a r k pinealocytes o f p o p u l a t i o n I as variants o f one population o f cells. The nucleus is large, oval, or polygonal (Fig. 1) and rarely lobulated (Fig. 15). M a n y Golgi complexes are usually widely dispersed in the vicinity of the nucleus. Each complex consists o f a system o f flattened sacs associated with a p o p u l a t i o n o f vesicles o f varying diameters. Some o f these vesicles are granular (GV), measuring

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50-300 nm in diameter (Fig. 9). GV are also observed throughout the cytoplasm of the cells. In the processes, however, and especially at their endings, the GV appear to be much larger, with a diameter of 300-1000 nm (Fig. 7) and are associated with numerous clear vesicles. This phenomenon of an enlargement of GV in the terminal of the process is especially conspicuous in the nulliparous females where, moreover, the number of GV in the endings of the pinealocyte processes of population I appears to be higher. In the pinealocytes of population I the appearance of the mitochondria is striking. Numerous microfilaments organized in one (Figs. 2, 9, 10, 15) or m a n y parallel bundles (Figs. 11, 14) are present in m a n y of the mitochondria. In some (Figs. 12, 14), only a portion of the matrix is occupied by the microfilament bundles, but in m a n y other mitochondria (Figs. 10, 11, 13, 14) the matrix is completely filled with this material. Single ribosomes, polyribosomes and cisternae of the granular endoplasmic reticulum are found scattered throughout the cytoplasm. Lipid droplets and lysosomal structures occur infrequently. Ciliated structures are found in some pinealocytes of population I (Figs. 18, 19). Often a concentric lamellar system is present near the cilia (Fig. 20), cross sections of which reveal a 9 + 0 arrangement of paired tubules (Fig. 21). Centrioles are also present, often associated with the ciliated structure. In some animals, large vacuoles associated with accumulations of glycogen granules are present in m a n y pinealocytes of population I (Fig. 16).

Pinealocytes o f Population H

The general appearance of the pinealocytes of population II is similar to that of population I and they are found throughout the pineal gland, not showing a preferred location. As in the pinealocytes of population I, the nucleus is large, oval or polygonal in shape. Lobulated nuclei, however, were never observed in the pinealocytes of type II. The structural differences between the nuclei of the two types of pinealocytes are only slight. In both cell types the chromatin is finely dispersed in the nuclear matrix, with some aggregation especially in a zone adjacent to the nuclear envelope. In general, however, the nucleus of pinealocytes of population II appears less electron dense than that of pinealocytes of population I.

Fig. 18. Ciliary derivative (cO in a pinealocyte of population I (P1), identified by the presence of a mitochondrion containing microfilaments(mit). Arrow centriole; asterisk material enclosedin the ciliary derivative. • 38,500 Fig. 19. Ciliary derivative (arrow) in a pinealocyte of population II (PH); g Golgi apparatus; double arrow centriole, x 40,000 Fig. 20. Lamellar system (arrows)located near a cilium (ci) in extracellular space; e centriole, x 21,600 Fig. 21. Cross section of a cilium in a pinealocyte of population I, displaying 9 paired tubules. A linear structure (arrow) is present between the paired tubules, x 40,000

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Table 2. Characteristics of the two populations of pinealocytes and of the glial cells Pinealocyte of population I

Pinealocyte of population II

Glial cell

Location

homogeneously dispersed in the parenchyma

homogeneously dispersed in the parenchyma

homogeneously dispersed in the parenchyma, but most frequently near a perivascular space

Nucleus

oval, polygonal or lobulated; chromatin finely dispersed with aggregations close to the nuclear envelope

oval or polygonal

oval

chromatin finely dispersed with aggregations close to the nuclear envelope

chromatin finely dispersed with aggregations close to the nuclear envelope

in general lighter than those of pinealocytes of population I

shape similar to that of nucleus of pinealocytes of population II

small vacuoles containing flocculent and moderately electron-dense material originating from the granular endoplasmic reticulum

large bundles of parallelrunning filaments in the cytoplasm

Perikaryon

granular vesicles

mitochondria containing bundles of microfilaments

numerous glycogen granules

The Golgi a p p a r a t u s and m i t o c h o n d r i a are usually arranged in the region o f the nucleus. Bundles of microfilaments (see mitochondria of pinealocytes of p o p u l a t i o n I), however, were never observed in the mitochondria o f pinealocytes of p o p u l a t i o n II. Moreover, granular vesicles originating from the saccules of the Golgi a p p a r a t u s (described in pinealocytes of population I) were not observed in the pinealocytes of p o p u l a t i o n II. Lipid droplets and lysosomal structures are c o m m o n in c o m p a r i s o n to their presence in the pinealocytes of population I. Ciliated structures were also found in some pinealocytes of population II (Fig. 19). The pinealocytes of population II are characterized by the presence of n u m e r o u s vacuoles containing flocculent material of m o d e r a t e electron density (Fig. 4), p r o b a b l y originating directly from the cisternae o f the granular endoplasmic reticulum. In some animals, large vacuoles associated with glycogen granules are present in pinealocytes of p o p u l a t i o n II (Fig. 17). Glial Cells Glial cells are sparsely scattered t h r o u g h o u t the parenchyma, Their general aspect resembles m o r e closely the pinealocytes o f population II, although they contain oval nuclei. They are readily identifiable due to the presence in the perikarya of large bundles of parallel-running filaments and n u m e r o u s glycogen granules (Figs. 5, 6). Table 2 summarizes these findings.

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265

Discussion

As in other mammals, one population of cells - here termed pinealocytes of population I - predominates in the pineal gland of the pipistrelle bat. In other mammals, these cells also predominate in number and are referred to variously as pinealocytes, light pinealocytes, parenchyma cells, clear pinealocytes, or light and dark chief cells (for details, see P6vet 1977, 1981), and are mainly characterized by the presence in their cytoplasm of granular vesicles originating from the G01gi saccules. From comparative phylogenetic and ontogenetic studies it is now apparent that these cells are derived phylogenetically from the neurosensory photoreceptor cells present in the pineal organ of submammalian vertebrates (Collin 1971; Oksche 1971 ; P~vet and Collin 1976; P~vet et al. 1977b) and that they belong to the sensory cell line as defined by Collin (1969, 1971). In consequence, these cells can be regarded as pinealocytes. Because some authors referred to all cells of the pineal organ as pinealocytes, Wolfe (1965) has termed these cells "pinealocytes s e n s u s t r i e t o " , a term that has subsequently been widely adopted by many authors. However, although such term is appropriate when only one population ofpinealocytes is present, it cannot be used for species in which two distinct populations have been observed. Consequently, in the pipistrelle, as in the mole-rat (Pbvet et al. 1976) and the noctule (P+vet et al. 1977a, b), we have termed these cells pinealocytes of population I. It should be realized that the term "pinealocyte s e n s u s t r i c t o " , used when only one population ofpinealocytes is present, and"pinealocytes of population I", used when more than one population of pinealocytes is present, characterize the same cells: the cells belonging to the sensory line as defined by Collin (1969, 1971) and containing granular vesicles. The question arises as to whether different types of pinealocytes s e n s u s t r i c t o (or of population I) can be distinguished. In the pipistrelle, light and dark pinealocytes of population I are morphologically distinguishable. Light and dark pinealocytes s e n s u s t r i c t o were also observed in numerous other mammals. Most authors are of the opinion that this difference in morphological aspect of these two types of pinealocytes is the result of a difference in the functional stage of the same cells (see details in P6vet 1981). This, however, does not imply that these two aspects are without physiological significance. In the pineal gland of the pipistrelle bat, in addition to the pinealocytes of population I and the glial cells, another population of cells has been observed which, due to the presence of ciliary derivatives and of a peculiar secretory process (see below), we call pinealocytes of population II. According to Collin (1969, 1971, 1979), the mammalian pineal gland is composed of two populations of intrinsic cells, pinealocytes and interstitial cells (also called glial, glial-like or astrocyte-like) cells. Collin also suggests that these two populations are unique due to their peculiar phylogeny. Pinealocytes are derived from photoreceptor cells, and interstitial cells from the so-called supportive cells of the pineal ol lower vertebrates. Consequently, according to Collin and his school, all cells of the mammalian pineal gland that are not pinealocytes s e n s u s t r i c t o are classified as interstitial (glial) cells. For example, Juillard (1979) classified the pinealocytes of population II that were described by us in the noctule bat (P6vet et al. 1977, b) as interstitial cells. If these pinealocytes of the noctule bat and similar cells of the mole-rat and pipistrelle bat were homologous to the supportive cells (cf. Juillard, 1979), it would be open to discussion which cell

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type could then be regarded as homologous with the glial cells also present in these species. Considering the diverse reports in the literature and our own observations, we feel that the concept put forth by Collin might be an oversimplification. As already discussed (P6vet 1977, 1981), the category of cells differing from pinealocytes sensu stricto (or population I) represents a heterogenous group, most often identifiable as interstitial, glial, or glial-like cells. In numerous mammals, however, another group of cells distinguishable from both glial cells and pinealocytes sensu stricto is present. These cells have been named pinealocytes of population II in the noctule bat (P~vet et al. 1977a, b), mole-rat (P~vet et al. 1976) and in the pipistrelle bat; pigment-containing cells in the chinchilla (Matsushima and Reiter 1975) and pocket gopher (Sheridan and Reiter 1973); and dark pinealocytes 1 in the rabbit (Wartenberg 1968; Romijn 1972), guinea pig (Lues 1971), macaque (Bererhi and Abbas-Terki 1970), and mouse (Upson et al. 1976). At present it is very difficult to establish whether these cells belong to the same functional group, but on the basis of the above-mentioned characteristics, it can be suggested that all these cells most probably belong to the same functional group (see details in P~vet 1977, 1981). It remains to be established whether the pinealocytes of population II belong to the cells of the sensory line as defined by Collin (1969) and can thus be considered to be true pinealocytes, or whether they are of a different phylogenetic origin. So far it can only be stated that in the mole-rat, noctule bat and pipistrelle bat, ciliary derivatives characterized, at least in the mole-rat and the noctule bat (P6vet et al. 1976, 1977b), by a 9+ 0 tubular pattern have been observed in this group of cells. In addition, in the noctule bat, a certain polarity in the distribution of cell organelles resembling that described in the rudimentary photoreceptor cells of nonmammalian vertebrate pineal organs, has also been reported. Moreover, these cells in the pipistrelle are characterized by the presence in their cytoplasm of vacuoles containing flocculent material of moderate electron density probably originating from cisternae of the granular endoplasmic reticulum. This peculiar secretory activity (ependymal-like secretory process; P6vet 1979) has been also observed in pinealocytes of population II of the mole-rat (P6vet et al. 1976) and noctule bat (P+vet et al. 1977a). Moreover, in species having only a single population of pinealocytes (garden dormouse, hedgehog, mole, rat), a similar ependymal-like secretory process is present in the pinealocytes sensu stricto (for details, see P6vet 1979, 1981). On the basis of these observations we have called these cells of the pipistrelle "pinealocytes of population Ir'; "pinealocytes" since we believe that they belong to the sensory cell line, and "population II" to differentiate them from pinealocytes of population I. The existence of two populations of true pinealocytes in some mammalian species is strongly supported by data in the literature. Flight (1975) and Meiniel (1979), for example, have described two different populations of photoreceptor cells in the pineal gland of Diemyctylus viridescens viridescens (Amphibia: Urodela) and Lampetra planeri (Agnatha: Petromizontidae). The presence in the same pineal organ of two morphologically as well as biochemically 1 It shouldbe realizedthat althoughthe terminologyusedis the same,thesedark pinealocytesare in no way related to the dark pinealocytessensu stricto (dark pinealocytesof population I)

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267

different populations of photoreceptors (Meiniel 1979, 1980; Meiniel and Hartwig 1980) points to the existence of two different lines of sensory cells (Meiniel 1980). This new concept of the phylogenetic evolution of the vertebrate pineal cells (for more details, see Meiniel 1980) would explain the presence of the two different populations of pinealocytes observed in some mammalian species. Additional evidence, however, does not support this concept. Petit (1976) and McNulty (1978a, b,c) described the presence of sensory cilia (9+0) in the supportive cells of some fish and reptiles. Moreover, McNulty (1978 a) described in the supportive cells an organelle-like specialization (glycogen-bound vacuoles) and noted that this was strikingly similar to that observed in the pinealocytes of population II of the noctule bat. However, such glycogen-bound vacuoles have also been observed in pinealocytes sensu stricto, for example, in the golden mole (Pbvet and Kuyper 1978); in the present study we have also found such a structure in the two populations of pinealocytes. Moreover, observing the illustrations presented by McNulty (1978c; especially Figs. 9, 10), it appears that the two cell types possessing a ciliary derivative with a 9+ 0 arrangement of microtubules, one of them being identified as a supportive cell by this author, could correspond to the two types of photoreceptors identified by Meiniel (1979, 1980) in Lampetraplaneri. Some recent studies on the glial cells, however, seem to indicate that this problem is more complex. Moller et al. (1978), using immunocytochemical techniques, have demonstrated that two glial marker proteins, GFA and S-100, are present in certain cells of the rat pineal, which could, thus, be classified as macroglial cells or cells of macroglial origin. Several factors indicate that these GFA- or S-100positive cells are the so-called interstitial cells of Wolfe (1965). These observations strongly suggest that some cells belonging to the interstitial/supportive cell line as defined by Collin (1969) and called interstitial, dark, glial or glial-like cells but also "true glial cells" could be present close to the pinealocytes in the parenchyma. Since there is considerable cytological and terminological confusion, this problem needs, in our opinion, thorough examination. In order to study ultrastructural changes of pinealocytes occurring throughout the annual reproductive cycle, pipistrelle bats were sacrificed at intervals during the course of the year. Noteworthy changes occur in the granular vesicles and the large vacuoles associated with accumulations of glycogen granules. Since intracellular glycogen is a source of energy and the glycogen content of the pineal is correlated with its activity (Kachi et al. 1973, 1974, 1975; Quay 1974), the abundance of glycogen granules in the pinealocytes of populations I and II of some animals may be related to changing synthetic and secretory activity. Although a large amount of glycogen has also been identified ultrastructurally in the mole-rat (P+vet et al. 1976), the golden-mole (P6vet and Kuyper 1978), the noctule bat (P6vet et al. 1977a) and the rabbit (Romijn et al. 1977), the exact functional significance of glycogen metabolism and its relationship with other biochemical activities of pinealocytes remain to be elucidated. As in the pipistrelle, glycogen granules in the noctule (P6vet et al. 1977a), the golden-mole (P6vet and Kuyper 1978) and also in the blind goby (McNulty 1978b) appear morphologically to be involved in the formation of intracellular vacuoles, which possibly represent the site of synthesis and/or maturation of secretory products (for details, see P6vet 1979, 1981). These structures have been observed in some pregnant female pipistrelles. Similarly, they

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have also been observed in a juvenile male killed in March, and in some adult males killed in April and May. We have thus been unable to correlate the presence of these formations to any phase of the annual cycle, and this peculiar structure is not characteristic of any of the reproductive classes studied (nulliparous, parous, pregnant and post-partum females, and juvenile, immature and adult males (for definitions, see Racey 1974). When present, however, these structures occur in numerous pinealocytes of both populations. Concerning the granular vesicles that most authors believe to represent a major secretory product in packaged form (for details and references, see P6vet 1979, 1981), the observations resulting from the present study can more easily be interpreted. The number of granular vesicles occurring in the terminals of pinealocytes of population I is higher in nulliparous females than in any other reproductive class examined. Since most female pipistrelles achieve sexual maturity in their first autumn, the nulliparous females examined in the present study were probably young animals born the previous summer (Racey 1974). Most nulliparous females examined were killed in May 1977 after two months of continuous torpor in an artificial hibernaculum. The observations may, therefore, be correlated either with age or with prolonged torpor. The fact that such an increase in number of granular vesicles was not so evident in a nulliparous female sacrificed one week after capture from its natural hibernaculum in March 1977 suggests that such vesicles may accumulate during continuous torpor in young females. However, in the three adult males killed in May 1977, which had also been in continuous torpor, an increase in the number of GV was not observed. Further studies are required to explain this phenomenon. Some secretory granules in the terminals of the pinealocyte processes are 2 to 3 times larger in diameter than the granular vesicles in the perikaryon. This means that during migration from the perikaryon to the ending of the process the secretory granules increase in size. This phenomenon, which is very evident in nulliparous females, was also present in all specimens studied. Such an increase in the diameter of granular vesicles, which, to our knowledge, has never been described in the pineal organ of other mammalian species, is difficult to explain. It is possible that large secretory granules are the result of a fusion of two or more small secretory granules. During our study, however, no evidence suggesting such a phenomenon has been obtained. Granular vesicles probably grow during transport by incorporation of new material or by a modification of the material present inside the vesicles. Concerning other cellular structures known to reflect an annual cycle, no convincing results have been obtained in the pipistrelle. Moreover, no change in the ependymal-like secretory process of the pinealocytes of population II has been observed. Although microcylinders have previously been described in mitochondria of rat pinealocytes (Lin 1965), the present report appears to be the first description of mitochondria containing bundles of microfilaments in pineal cells. McNulty (1978c) has described in the photoreceptor cells of the troglobytic fish a mitochondrion "which is nearly filled with crystalline inclusion" similar to that observed in the pipistrelle. In the pipistrelle, mitochondria containing microfilaments occur frequently. Intramitochondrial crystals, paracrystalline inclusions or

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microfilaments have also been observed in normal and pathological tissues (see list and reference in Ghadially 1975). The chemical nature of such intramitochondrial inclusions is at present conjectural, but it is generally assumed that they are protein in nature (see Ghadially 1975). In the pipistrelle bat it seems likely that these mitochondria containing microfilaments are intrinsic components of pipistrelle pinealocytes of population I, and that they may represent products of (unusual?) mitochondrial activity. At the present time, however, the functional significance of these microfilaments remains elusive. References Bererhi A, Abbas-Terki M (1970) Structure fine de l'6piphyse du Magot d'Alg6rie (Macacus sylvanus L.) Bull Assoc Anat 148: 285-294 Bozza G (1927) Contributo alla conoscenza dello sviluppo della regione epifisaria in alcuni mammiferi compreso l'uomo. Arch Ital Anat Embriol 24:532-626 Collin JP (1969) Contribution fi l'~tude de l'organe pin~ale. De l'~piphyse sensorielle fi la glande pin~ale: modalit6s de transformation et implications fonctionelles. Ann Stat Biol De Besse-en-Chandesse, Suppl. 1:1-359 Collin JP (1971) Differentiation and regression of the cells of the sensory line in the epiphysis cerebri. In: Wolstenholme GEW, Knight J (eds) The pineal gland (a CIBA symposium). Churchill Livingstone, Edinburgh London, pp 79-125 Collin JP (1979) Recent advances in pineal cytochemistry. Evidence of the production of indoleamines and proteinaceous substances by rudimentary photoreceptor cells and pinealocytes of Amniota. In: Ari~ns Kappers J, P6vet P (eds) The pineal gland of vertebrates including man. Progr Brain Res Vol 52. Elsevier, Amsterdam, pp 271-296 Creutzfeldt HG (1912) 13ber das Fehlen des Epiphysis cerebri bei einigen S/iugern. Anat Anz 42:517-521 Flight WFG (1975) On the pineal of the urodele, Diemictylus viridescens viridescens. Thesis. Utrecht Ghadially FN (1975) Ultrastructural pathology of the cell. Butterworths, London Glauert AM, Glauert RH (1958) Araldite as an embedding medium for electron microscopy. J Biophys Biochem Cytol 4:191-194 Juillard MT (1979) The proteinaceous content and possible physiological significance of dense-cored vesicles in hamster and mouse pinealocytes. Ann Biol Anim Biochim Biophys 19:413-428 Kachi T, Matsushima S, Ito T (1973) Diurnal variations in pineal glycogen content during the oestrous cycle in female mice. Arch Histol Jpn 35:153-159 Kachi T, Matsushima S, Ito T (1974) Effect of continuous darkness on diurnal rhythm in glycogen content in pineal cells of the mouse: a semi-quantitative histochemical study. Anat Rec 179: 405-410 Kachi T, Matsushima S, Ito T (1975) Postnatal observations on the diurnal rhythm and the lightresponsiveness in the pineal glycogen content in mice. Anat Rec 183:39-46 Kappers J Arifins (1976) The mammalian pineal gland, a survey. Acta Neurochir 34:109-149 Krabbe KH (1920) Bidrag til kundsgaben om corpus pineale hos pattedyrene. KG1 Dan Vidensk Selsk, Biol Nedd 2:1-126 Lin HS (1965) Microcylinders within mitochondrial cristae in the rat pinealocyte. J Cell Bio125:435-441 Lues G (1971) Die Feinstruktur der Zirbeldr/ise normaler, tr~ichtiger und experimentell beeinfluBter Meerschweinchen. Z Zellforsch 114:38-60 Luna E (1921) Morfogenesi dei centri nervosi nei chirotteri. Parte I. Le prime fasi di sviluppo dei centri nervosi in Rhinolophus hipposideros (Bechstein). Ric Morfol 2:1-102 Matsushima S, Reiter RJ (1975) Comparative ultrastructural studies of the pineal gland of rodents. In: Hess M (ed) Electron microscopic concepts of secretion: ultrastructure of endocrine and reproductive organs. John Wiley & Sons, New York, pp 335-356 McNulty JA (1978 a) The pineal of the troglophilic fish, Chologasteragassizi: an ultrastructural study. J Neural Transm 43:47-71 McNulty JA (1978 b) A light and electron microscopic study of the pineal in the blind goby, Typhlogobius californiensis (Pisces: Gobiidae). J Comp Neurol 181:197-211 McNulty JA (1978c) Fine structure of the pineal organ in the troglobytic fish, Typhliehthyes subterraneous (Pisces: Amblyopsidae). Cell Tissue Res 195: 535-545

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Meiniel A (1979) Detection and localization of biogenic amines in the pineal complex of Lampetra planeri (Petromyzontidae). In: Ari~ns Kappers J, P~vet P (eds) The pineal gland in vertebrates including man. Progr Brain Res, Vol 52. Elsevier, Amsterdam, pp 303-307 Meiniel A (1980) Ultrastructure of serotonin-containing cells in the pineal organ of Lampetra planeri (Petromyzontidae). A second sensory cell line from photoreceptor cell to pinealocyte. Cell Tissue Res 207:407-427 Meiniel A, Hartwig HG (1980) Indoleamines in the pineal complex of Lampetra planeri (Petromyzontidae). A fluorescence microscopic and microspectrofluorometric study. J Neural Transm 48: 65-84 Moller M, Ingild A, Bock E (1978) Immunohistochemical demonstration of S-100 protein and GFA protein in interstitial cells of rat pineal gland. Brain Res 140:1-13 Oksche A (1971) Sensory and glandular elements of the pineal organ. In: Wolstenholme GEW, Knight J (eds) The pineal gland. A CIBA Foundation Symposium (1970). Livingstone Churchill, Edinburgh London, pp 127-146 Petit A (1976) Contribution ~il'6tude de l'6piphyse des reptiles: le complexe 6piphysaire des lacertiliens et l'+piphyse des ophidiens. Etude embryologique, structurale, ultrastructurale; analyse qualitative et quantitative de la s+rotonin dans les conditions normales et exp+rimentales. Th~se, Strasbourg P+vet P (1976) Correlations between pineal gland and sexual cycle. An electron microscopical and histochemical investigation on the pineal gland of the hedgehog, mole, mole-rat and white rat. Thesis, Amsterdam P+vet P (1977) On the presence of different populations of pinealocytes in the mammalian pineal gland. J Neural Transm 40:289-304 P+vet P (1979) Secretory process in the mammalian pinealocyte under natural and experimental conditions. In: Ari~ns Kappers J, P~vet P (eds) The pineal gland of vertebrates including man. Progr Brain Res, Vol 52. Elsevier, Amsterdam, pp 149-194 P~vet P (1981) Ultrastructure of the mammalian pinealocytes. In: Reiter RJ (ed) The pineal: anatomy and biochemistry. CRC Press, Palm Beach, USA, in press P~vet P, Collin JP (1976) Les pin6alocytes de mammif6re: diversit+, homologies, origine. Etude chez la taupe adulte (Talpa europaea L.). J Ultrastruct Res 57:22-31 P6vet P, Kuyper MA (1978) The ultrastructure of pinealocytes in the golden mole (Amblysomus hottentotus) with special reference to the granular vesicles. Cell Tissue Res 191 : 39-56 P+vet P, Kappers J Ari~ns, Nevo E (1976) The pineal gland of the mole-rat (Spalax ehrenbergi, Nehring). I. The fine structure of pinealocytes. Cell Tissue Res 174:1-24 P6vet P, Kappers J Ari~ns, Vofite M (1977a) The pineal gland of nocturnal mammals. I. The pinealocytes of the bat Nyctalus noctula Schreber. J Neural Transm 40:47-68 P6vet P, Kappers J Ari6ns, Vofite AM (1977b) Morphologic evidence for differentiation of pinealocytes from photoreceptor cells in the adult noctule bat (Nyctalus noetula Schreber). Cell Tissue Res 182:99-109 Quay WB (1970) Pineal organ. In: Biology of bats, Vol 2. Academic Press, New York, pp 311-318 Quay WB (1974) Pineal chemistry in cellular and physiological mechanisms. C. Thomas, Springfield, Ill Quay WB (1976) Seasonal cycle and physiological correlates of pinealocyte nuclear and nucleolar diameters in the bats, Myotis lucifugus and Myotis sodalis. Gen Comp Endocrinol 29:369-375 Racey PA (1969) Diagnosis of pregnancy and experimental extension of gestation in the pipistrelle bat, Pipistrellus pipistrellus. J Reprod Fertil (Suppl) 19:465-474 Racey PA (1974) Ageing and assessment of reproductive status of pipistrelle bats, Pipistrelluspipistrellus. J Zool Lond 173:264-271 Racey PA (1979) The prolonged storage and survival of spermatozoa in Chiroptera. J Reprod Fertil (Suppl) 56:391-402 Racey PA, Tam WH (1974) Reproduction in male Pipistrellus pipistrellus (Mammalia, Chiroptera). J Zool Lond 172:101 122 Reiter RJ (1973) Pineal control of a seasonal reproductive rhythm in male golden hamsters exposed to natural daylight and temperature. Endocrinology 92 (2):423-430 Reiter RJ (1975) Exogenous and endogenous control of the annual reproductive cycle in the male golden hamster: participation of the pineal gland. J Exp Zool 191:111-120 Reiter RJ (1978) Interaction of photoperiod, pineal and seasonal reproduction as exemplified by findings in the hamster. Prog Reprod Biol 4:169-190

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Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:20~212 Romijn H (1972) Structure and innervation of the pineal gland of the rabbit, Oryctolagus cuniculus L., with some functional considerations. Thesis, Free Univ Amsterdam Romijn HJ, Mud MT, Wolters PS (1977) Electron microscopic evidence of glycogen storage in the dark pinealocytes of the rabbit pineal gland. J Neural Transm 38:231-237 Sheridan MN, Reiter R.J (1973) The fine structure of the pineal gland in the pocket gopher (Geomys bursarius L.). Am J Anat 136:363-382 Stammer A (1972) Studies on the pineal organ of the great horse-shoe bat (Rhinolophusferrumequinum). Gen Comp Endocrinol 18:624 Upson RM, Benson B, Satterfield V (1976) Quantification of ultrastructural changes in the mouse pineal in response to continuous illumination. Anat Rec 184:311-324 Venable JM, Coggeshall W (1965) Simplified lead stain for use in electron microscopy. J Cell Biol 25: 407-408 Vivien-Roels B (1980) Activit6 sexuelle et glande pin6ale. La Recherche 113:833-835 Wartenberg H (1968) The mammalian pineal organ: electron microscopic studies on the fine structure of pinealocytes, glial cells and on the perivascular compartment. Z Zellforsch 86:74-97 Wolfe DE (1965) The epiphyseal cell: An electron-microscopic study of its intercellular relationship and intracellular morphology in the pineal body of the albino rat. In: Arifins Kappers J (ed) Structure and function of the epiphysis cerebri. Progr Brain Res 10, Elsevier, Amsterdam, pp 332-376 Accepted December 5, 1980