Cell Tiss. Res. 159, 303--309 (1975) �9 by Springer-Verlag 1975

Vacuolated Pinealocytes in the Hedgehog (Erinaceus europaeus L.) and the Mole (Talpa europaea L.)

Paul Pevet *

Netherlands Central Institute for Brain Research, Amsterdam, the Netherlands (Director: Prof. Dr. J. Arifins Kappers)

Received November 18, 1974

Summary. Special cells in the hedgehog and mole pineal gland which are characterized by an enormous single vacuole were studied. The presence of secretory granules (in the hedgehog) and of paracrystalline structures (in the mole) in the cytoplasm of these cells indicates that they are pinealocytes.

The nature and the role of the phenomenon of vacuolization are discussed.

Key words: Pinealocytes - - Vacuole - - Hedgehog - - Mole.

Rdsumd. Des eellules particuli~res de la glande pin6ale du H6risson et de la Taupe, caract6ris6es par la pr6sence d'une vacuole ~norme, ont 6t6 6tudi6es. La pr6sence dans le cytoplasme de ces cellules de grains de s~cr6tion (chez le H6risson) et de structures para- eristallines (chez la Taupe) permet d'identifier ces cellules comme 6rant des pin6alocytes.

La nature et le r61e de ce ph6nom~ne de vacuolisation sont discutCs.

Introduction

I n view of the fact t ha t the pineal g land controls the sexual cycle of mammals (Reiter, 1973), it seems of interest to examine the pineal gland of wild mammals l iving under na tu ra l l ighting condit ions and showing a characteristic sexual cycle.

I n previous invest igat ions we have studied the ultrastIxlcture of pinealocytes of two wild mammals : the hedgehog and the mole (Pevet, 1972, 1975; Pevet and Saboureau, 1973). The u l t ras t ruc tura l changes of the hedgehog pinealocyte were also followed during different phases of the sexual cycle (Pevet, 1972, 1975; Peve t and Saboureau, 1972, 1973). I n the pineal of these animals, some cells, characterized by the presence of a single enormous vacuole, were observed.

The present paper is concerned with the identif icat ion and the s tudy of these rare bu t special cells.

Material and Methods

All animals used in this investigation were previously used in the study of normal hedgehog and mole pinealocytes (Pevet, 1972, 1974; Pevet and Saboureau, 1973). They were decapitated under anesthesia; their pineal glands were fixed in glutaraldehyde 2.5%-phosphate buffer

Send o//print requests to: Dr. Paul Pevet, Netherlands Central Institute for Brain Research, Ijdijk 28, Amsterdam-O, The Netherlands. * Acknowledgements. The author wishes to thank Dr. A. R. Smith and Prof. Dr. J. Ari~ns Kappers for their help in the present work and for the revision of the paper, and Miss. J. Sels for typing the manuscript.

304 P. Pevet

0.2 M; pH 7.25. After washing the organs in buffer, they were placed in a post-fixative (OsOa-phosphate buffer 0.2 M; pH 7.25; room temperature). The glands were then dehydrated and embedded in araldite 1 (for details see Pevet, 1974, and Pevet and Saboureau, 1973).

Ultrathin sections were cut on an LKB ultramicrotome with a glass knife, made with an LKB knife maker. The sections were stained with uranyl acetate and lead citrate (Reynolds, 1963; Venable and Goggeshall, 1965). They were observed under a Philips 200 electron microscope.

Results

The cells descr ibed here have been observed only ve ry rare ly in the hedgehog and mole pineal glands s tud ied up ti l l now (20 male and female moles, 83 male hedgehogs). Nevertheless , a few were seen in p rac t ica l ly all the pineals s tudied. However , in one pineal of a male hedgehog, and especial ly in two pineal g lands of female moles, a large number of these cells were found.

The cells are charac ter ized by the presence of a single huge vacuole (Figs. 1-3) which a lmos t comple te ly occupies the cellular body. A thin s t r ip of cy top lasm (Figs. 1-3) is stil l present , and the nucleus is s i tua ted a t the cellular pe r iphe ry (Figs. 1-3).

Only one large vacuole per cell was ever seen. Very of ten m a n y small vacuoles s i t ua t ed near the large one (Figs. 2, 7), were seen to fuse wi th i t (Fig. 7). An exchange of p roduc t s be tween cy top lasm and vacuole is also appa ren t (Figs. 3-5).

I n the nucleus of these cells, the chromat in is widely dispersed in the nucleo- p l a sm; a few chromat in concentra t ions are located in an area adjoin ing the nuc lear envelope (Figs. 1-3, 5). These features are character is t ic of the nucleus of these special cells and of the nucleus of hedgehog and mole p inealocytes in genera l (Pevet , 1972, 1974a, 1974b; Peve t and Saboureau, 1973).

I n the remain ing cy top lasm all the usual cell organelles were observed, such as numerous r e l a t ive ly large mi tochondr ia (Figs. 1-4, 7), r ibosomes (Figs. 1-5), g r anu la r endoplasmic re t icu lum (especially in the mole cell) (Fig. 4) and Golgi complexes (Figs. 1, 3, 4). Moreover, in the hedgehog pineal cells, m a n y secre tory g ranu la r vesicles could be d is t inguished (Figs. 6, 7). In the vacuola ted mole p inea l cells some parac rys ta l l ine s t ruc tures were also present (Fig. 4).

Discussion

The presence of secre tory granules is t yp ica l for hedgehog pinealocytes (Pevet , 1972, 1975; Peve t and Saboureau, 1973) while the paracrys ta l l ine s t ruc tures are character is t ic of those of the mole (Pevet , 1974). As these two

Fig. 1. Vacuolated pinealocyte of a male hedgehog. The vacuole (*) is large but does not occupy the cell completely. Lipids (llp), Golgi complex (9), mitechondria (mit) are present

in the remaining cytoplasm; n nucleus of vacuolated pinealocyte, x 15300 Fig. 2. Vacuolated pinealocyte of a male hedgehog. The vacuole (*) practically occupies the entire cell. The nucleus (n) is pressed against the cell periphery, while merely a thin strip of

cytoplasm remains. P normal pinealocyte, v small vacuole. •

1 Part of the hedgehogs and all of the moles were sacrificed and the pineal glands fixed and embedded in the Laboratoire de Physiologic Compar6e and SGMEAB, Universit6 de Poitiers, France. The other hedgehogs were sacrificed in the Centre d'6tndes biologiques des animaux sauvages, Beauvoir sur Niort, :France.

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306 P. Pevet

Fig. 3. Vacuol~ted pinealocyte of a female mo]e. The vacuole (*) occupies the entire ceil. The nucleus (n) lies against the cell periphery. A Golgi complex (g) and mitochondria (mit) are observed in the thin strip of the remaining cytoplasm. The arrow points to exchange of products

between cytoplasm and vacuole. P normal pinealocyte. • 8000 Fig. 4. Larger magnification of part of the remaining cytoplasm of a vacuolated male mole pinealocyte. Mitochondria (mit), Golgi complex (g), granular endoplasmic retieulum (r) and a paracrystalline structure (pcs) next to a mitochoudrion are present. In an adjacent pinealo- cyte (P) a paracrystalline structure is also seen (pcs'). n Nucleus of the vacuolated pinealo-

cyte, (*) vacuole. •

Vacuolated Insectivore Pinealocytes 307

Fig. 5. Larger magnification of part of the remaining cytoplasm in a vaeuolated pinealocyte from a male hedgehog. The arrows point to an exchange of products between cytoplasm and vacuole (*). n Nucleus of the vacuolated pinealoeyte, gs secretory granule. • Fig. 6. Larger magnification of part of the remaining cytoplasm in a vacuolated pinealocyte from a male hedgehog. Secretory granules (gs) are present in the cytoplasm; (*) vacuole.

• 26550 Fig. 7. Larger magnification of part of the cytoplasm in a vacuolated pinealocyte from a male hedgehog. Many small vacuoles (v) and secretory granules (gs) are present in the cyto- plasm. The small vacuoles appear to fuse (v') with the large vacuole (*); mi t mitochondria.

• 17800

organelles are present in the cy top lasm of the vacuo la ted cells descr ibed here i t is l ike ly t h a t t h e y represen t p inealocytes .

This t y p e of p inea locyte is charac te r ized b y the fo rma t ion of a single ve ry large vacuole which a lmos t occupies the cell complete ly . I t seems t h a t th is vacuole is formed b y the increase in size of a p r i m a r y vacuole b y i ts confluence wi th smal ler vacuoles of ten observed nex t to the large one. So far, we could no t de te rmine the origin of e i ther the small vacuoles or of the p r i m a r y vacuole.

The na tu re of the large vacuoles in p inealocytes , f i rs t descr ibed here, cannot be easi ly expla ined. The vacuo la t ion of the p inea locy te m a y be due to i ts de- generat ion. This could expla in t he ex t reme scarc i ty of these special cells in the

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308 P. Pevet

m a j o r i t y of the animals s tudied. Remarkab ly , however, these cells show the same aspect as LH-cel ls in the adenohypophys i s of male ra ts and of h a m s t e r af ter long- te rm cas t ra t ion (Costoff, 1973 ; Girod and Dubois, 1965). In the rat , the LH-b lood level increases for one week af ter o rch idec tomy and then remains a t a con- s t an t level (McCann, 1965), while in the adenohypophys i s the LH-concen t r a t ion cont inues to increase, depending on the t ime of cas t ra t ion (McCann, 1965). Af te r long- te rm cas t ra t ion, the vacuola t ion of LH-cel ls in the adenohypophys i s cor- responds to a hormonal re ten t ion of L H in the cell. The fact t h a t m a n y vacuola ted p inea locy tes are observed in some animals (especially in the female mole) and the fac t t h a t dur ing vacuola t ion t hey show a normal secretory process po in t to the poss ib i l i ty t h a t ~ecretory compounds p roduced by the cy top lasm of the p inea locy tes are s tored in the vacuoles. I n this connection, i t is in teres t ing to note t h a t in the vacuo la ted p inealocytes of the hedgehog, m a n y more secre tory granules are present t han in pinealocytes having not such a large vacuole. This observa t ion could po in t to an increased product ion of secre tory granules in p inea locy tes conta in ing such huge vacuoles.

On the o ther hand, i t might be t h a t the large concentra t ion of secre tory granules in the remain ing shell of cy top lasm is mere ly due to thei r accumula t ion in a smal ler space t han is ava i lab le in p inealocytes wi thout such a s t ructure .

I t would be in teres t ing to inves t iga te whether there is any re la t ion between the appea rance of vacuo la ted p inea locytes and changes in the physiological condi t ion of the animals s tudied.

Prev ious au thors (Allanson, 1934; Courrier, 1927; Gerry, 1969; Godet , 1949; Gougeon, 1972; Peyre , 1968 and Saboureau, 1969) have observed t h a t large var ia t ions in the hormonal equi l ibr ium of the mole and hedgehog occur dur ing the seasonal sexual cycles. I n par t icu lar , the hormone product ion of the adeno- hypophyseo -gonada l axis is affected.

I n a fur ther s t u d y an a t t e m p t will be made to inves t iga te a possible correlat ion be tween the vacuola t ion of p inealocytes in these two m a m m a l i a n species and a specific phase of thei r sexual cycle.

References

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Costoff, A. : Ultrastructure of rat adenohypophysis. Correlation with function. New York and London: Academic Press 1973

Courrier, R. : Etude sur le d6terminisme des caract~res sexuels secondaires chez quelques Mammif~res s l'activit6 testiculaire p6riodique. Arch. Biol. (Libge) 37, 173-334 (1927)

Gerry, H. : Contribution s l'6tude de la cytologie ant6hypophysaire. (Recherches chez un Mammif6re hibernant.) Th6se (Lyon) (1969)

Girod, C., Dubois, P. : Etude ultrastrueturale des eellules gonadotropes ant6hypophysaires, chez le Hamster dot6 (Mesocricetus auratus Waterh.). J. Ultrastruct. Res. 13, 212-232 (1965)

Godet, R.: Recherches d'anatomie, d'embryologie normale et exp6rimentale sur l'appareil g6nital de la Taupe (Talpa europaea L.). Bull. Biol. France et Belg. 83, 25-296 (1949)

Gougeon, A. : Analyse histologique et histoenzyraologique de l'interstitielle ovarienne chez deux Inseetivores Europ~ens: la Taupe (Talpa europaea) et le H6risson (Erinaceus europaeus). Th~se IIIe cycle. Universit6 de Poitiers (1972)

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McCann, S. M. : Gonadotrophic functions of the adenohypophysis. In : Physiological controls and regulations. W. S. u and J. R. Brobeck, eds., p. 135-137. Philadelphia: W. B. Saunders Comp. 1965

Pevet, P. : E tude ul t ras t ructurale de l '6piphyse du H6risson male. Evolut ion en fonction du cycle sexuel. Th~se I I I e cycle. Universit6 de Poitiers (1972)

Pevet, P.: The pineal gland of the mole (Talpa europaea L.). I. The fine s t ructure of the pinealocytes. Cell Tiss. Res. 153, 277-292 (1974)

Pevet, P. : E tude strueturale et ul t ras t ructurale de l '6piphyse du H6risson m&le (Erinaceus europaeus L.). Evolut ion en fonction du cycle sexuel. Vme entretiens de Chiz6. Problbmes endoeriniens chez les Mammif6res sauvages - - Aspects mbtaboliques et 6cophysiologiques, 11-13 octobre 1973 (1975) (in press)

Pevet, P., Saboureau, M. : Modifications ul t rastructurales dans l '6piphyse du H6risson m&le au cours du repos sexuel. Congr6s de la Soc. Europ. d 'Endocr. Comp. Montpellier 1971. Gen. eomp. Endocr. 18, 3 (1972)

Pevet, P., Saboureau, M.: L'6piphyse du H6risson (Erinaceus europaeus L.) male. 1) Les pin~aloeytes et leur variat ions ul t rastructurales consid~r~es au cours du cycle sexuel. Z. Zellforsch. 143, 367-385 (1973)

Peyre, A. : Cycles g~nitaux et correlation hypophyso-g~nitale chez trois Insectivores Europ~ens. In : Cycles g~nitaux saisonniers de Mammif~res sauvages, p. 133-149. Paris: Masson & Cie. 1968

Reiter, R. J . : Pineal control of a seasonal reproductive r h y t h m in male golden hamsters exposed to natural dayl ight and temperature. Endocrinology 92, 2, 4 2 3 4 3 0 (1973)

Reynolds, E. S. : The use of lead citrate at high p i t as an electron-opaque s tain in electron microscopy. J . Cell Biol. 17, 208-212 (1963)

Saboureau, M.: Quelques nouveaux crit6res biochimiques de l 'activit6 g6nitale ehez le tt6risson male. Th6se I I I e cycle, Universit6 de Poitiers (1969)

Venable, J . M., Coggeshall, W. : Simplified lead stain for use in electron microscopy. J . Cell Biol. 25, 407-408 (1965)

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