Cell and Tissue Research
Cell Tiss. Res. 182, 215-219 (1977)
9 by Springer-Verlag 1977
The Pineal Gland of the Mole (Talpa europaea L.) IV. Effect of Pronase on Material Present in Cisternae of the Granular Endoplasmic Reticulum of Pinealocytes P. Pevet Netherlands Institute for Brain Research, Amsterdam, The Netherlands
Summary. The effect of a proteolytic enzyme, pronase, on material present in cisternae of the granular endoplasmic reticulum of mole pinealocytes demonstrates their proteinaceous nature. Key words: Pinealocyte - Mole Proteinaceous material - Pronase.
Granular endoplasmic reticulum -
R6snm6. L'6tude des effets de l'action d'une enzyme prot6olytique, la pronase, sur les accumulations de mat6riel pr6sentes dans les citernes du r6ticulum endoplasmique granulaire des pin6alocytes de la Taupe, a permis de d6montrer leur nature prot6ique.
Introduction Accumulation of material in single cisternae of the granular endoplasmic reticulum (GER) or between the two layers of the nuclear envelope, has been observed in some pinealocytes of the dormouse (Roux et al., 1974; Roux and Richoux, 1975), the mole (Pevet, 1974, 1976; Pevet and Smith, 1975), the mole-rat (Pevet, 1976; Pevet et al., 1976) and some pineal parenchymal cells of the parakeet (type II, Quay et al., 1968). Observations reported earlier (see Pevet, 1976, 1977) led to the hypothesis that this material represents an exacerbation of a secretory process present in the pinealocytes of numerous mammals and non-mammals, the secretory process probably being involved in the synthesis of an antigonadotropic compound(s) (see details in Pevet, 1976, 1977). Similar accumulation of material has also been observed in several other cell types (see Fawcett, 1966) and was assumed to consist of protein (Fawcett, 1966; Cresti et al., 1974). In tissue embedded in epoxy resins, Monneron (1966) and Monneron and Bernhard (1966) have demonstrated that proteinaceous inclusions in pancreatic Send offprint requests to: Dr. P. Pevet, Netherlands Institute for Brain Research, IJdijk 28, Amsterdam-O, The Netherlands Acknowledgements: The author wishes to thank Prof. Dr. J. Ari~ns Kappers for his help in the present work and for revision of the manuscript, Miss M.T. Mud and Mr. P.S. Wolters for their skillful assistance and Mrs. M.M. Smidt for typing the manuscript
216
P. Pevet
cells o r in a d e n o h y p o p h y s i a l cells a r e d i g e s t e d b y p r o t e o l y t i c e n z y m e s s u c h as pronase and pepsin. In order to investigate the eventual proteinaceous nature o f t h e m a t e r i a l o b s e r v e d in t h e c i s t e r n a e o f t h e G E R o f s o m e p i n e a l o c y t e s , p r o t e o l y t i c e n z y m e s w e r e e m p l o y e d in t h e p r e s e n t study. T h e m o l e (Talpa europaea L.) w a s c h o s e n b e c a u s e t h e m a j o r i t y o f t h e m a t e r i a l a c c u m u l a t e d in t h e G E R o f this a n i m a l s h o w a p a r a c r y s t a l l i n e s t r u c t u r e (Pevet, 1974, 1976; P e v e t a n d S m i t h , 1975), w h i c h is r e a d i l y i d e n t i f i a b l e . P r o n a s e is a p o w e r f u l p r o t e o l y t i c e n z y m e w h i c h h y d r o l y s e s a l m o s t all p e p t i d e b o n d s in p r o t e i n s ( M o n n e r o n , 1966) a n d t h e r e f o r e a p p e a r e d to be a n a p p r o p r i a t e c h o i c e in t h e s e i n v e s t i g a t i o n s .
Material and Methods All four moles (3 females and 1 male) used in this study were captured in Deux-S~vres (France) in January, 1977. The animals were decapitated under ether anesthesia; the skull was opened and the region of the pineal flooded with cold fixative (2.5~ glutaraldehyde in 0.1 M phosphate buffer, pH7.25 at 4~ After its removal, the gland was placed in the same fixative at 4~ for 1 h. After washing in phosphate buffer for 12 h, the organs were dehydrated (without post-fixation) and embedded in araldite (Glauert and Glauert, 1958). Ultrathin sections were cut on a LKB ultramicrotome with glass knives and then collected on gold grids.
Enzymatic Extractions. The sections were oxidized for 30 min in a 10~ periodic acid solution, rinsed several times with distilled water and then floated for 30min, 1, 4 or 24h at 40~ on an enzymatic solution containing 0.5~o pronase (Koch-Light Laboratories Ltd, 45000 P.U.K./g) in distilled water, pH 7.4 (adjusted with NaOH 0.01 N). For controls, the last bath did not contain pronase. After enzymatic extraction, the sections were washed several times with distilled water and stained with uranyl acetate and lead citrate (Reynolds, 1963; Venable and GoggeshaU, 1965). Observations were made with a Philips 200 electron microscope.
Results A f t e r 30 a n d 60 m i n o f e n z y m a t i c t r e a t m e n t , t h e t y p i c a l p a r a c r y s t a l l i n e s t r u c t u r e o f t h e m a t e r i a l a c c u m u l a t e d in t h e G E R was l a r g e l y i n t a c t , i n d i c a t i n g t h a t t h e p r o n a s e h a d little effect at t h e s e t i m e p e r i o d s . H o w e v e r , in a few p i n e a l o c y t e s a v i s i b l e d i g e s t i o n h a d t a k e n place.
Fig. l a-e. Normal mole pinealocyte and pinealocytes after four hours treatment with pronase. a Control section. Accumulation of material (arrows) showing a typical paracrystalline structure between the two layers of the nuclear envelope, x 31,100. b Control section. Material (arrows) accumulated in the cisternae of the GER showing a typical paracrystalline structure, x 26,000. e Control section. Accumulation of material between the two layers of the nuclear envelope (arrow) without apparent paracrystalline organization, x 59,000. d Section treated with pronase. Two accumulations of material (*) located in cisternae of the GER are digested by pronase. The arrow points to remains of the paracrystalline structure, the hydrolysis of which was incomplete, x 28,900. e Section treated with pronase. Two digested accumulations of material (*). In the material located between the two layers of the nuclear envelope, hydrolysis of the paracrystalline structure is incomplete (arrow). mit Mitochondria, n nucleus, P pinealocyte, x 39,000 Fig. 2. Control section. A granular vesicle (arrow) present near the Golgi apparatus (g) is observed in a pinealocyte; n nucleus, x 40,000
Effect of Pronase on Mole Pinealocytes
217
218
P. Pevet
Practically all accumulations were digested after four hours of enzymatic treatment (Fig. 1). Only in a few rare cases were fragments of paracrystalline structure still observed (Fig. 1), the hydrolysis of which was evidently incomplete. After twenty four hours of treatment, all accumulations appeared to have been digested by pronase. However, the general cellular morphology was not well preserved. In some control sections, granular vesicles were observed (Fig. 2). Such vesicles were never seen in the enzyme-treated sections. Discussion
The cytochemical results obtained in this study prove that the material accumulated in the cisternae of the GER or between the two layers of the nuclear envelope of mole pinealocytes is proteinaceous in nature. This result justifies the term "accumulation o f proteinaceous material" earlier used for these structures (Pevet, 1976, 1977) and may point to their physiological importance (see Pevet, 1977). From the observations that paracrystalline structures remain intact after 30 and 60 min of enzymatic treatment but evidence of digestion is visible suggests that additional proteinaceous material is also present and possesses a greater sensitivity to pronase. In previous investigations (Pevet, 1974, 1976; Pevet and Smith, 1975), proteinaceous material has been observed which lacks a paracrystalline organization. It seems that the material digested by the pronase during the first hour corresponds to that having no paracrystalline structure as judged from the observation of structures with exclusively paracrystalline organization after 30 and 60 rain of enzymatic treatment. Nevertheless, this observation does not permit the conclusion that the different types of proteinaceous material observed in the mole are different in nature. Other studies will be necessary to solve this problem. We do believe that these different types might represent only different developmental stages of the same formation (see Fig. 21 in Pevet, 1974). Another point of interest is the nature of the matrix of granular vesicles originating from the Golgi apparatus. The fact that these vesicles were observed in control sections, but not in those subjected to pronase may prove that their proteinaceous matrix had been digested by pronase. This would agree with the results of Collin and Meiniel (1972), Vivien-Roels (1976) and Petit (1976) who, using the same technique, demonstrated the proteinaceous nature of the matrix of the granular vesicles in the pineal cells of lacertilians, magpies, tortoises and snakes ( N a t r i x n a t r i x ) . It can not be stated with certainty, however, that the matrix of the granular vesicles is actually digested by this enzyme. The granular vesicles are extremely scarce in normal mole pinealocytes (Pevet, 1974, 1976), so that positive identification of possibly digested granular vesicles in enzymatically treated sections is very difficult. References Collin, J.P., Meiniel, A.: L'organe pin6al du genre Lacerta (Reptile, lacertilien): action d'enzymes protrolytiques sur les grains denses (500-3400 A) des photorrcepteurs rudimentaires. C.R. Soc. Biol. (Paris) 166, 370-380 (1972)
Effect of Pronase on Mole Pinealocytes
219
Cresti, M., Pacini, E., Simoncioli, C.: Uncommon paracrystalline structures formed in the endoplasmic reticulum of the integumentary cells ofDiplotaxis erucoides ovules. J. Ultrastruct. Res. 49, 218-223 (1974) Fawcett, D.W.: An atlas of fine structure: The cell. Philadelphia and London: Saunders Co. 1966 Glauert, A.M., Glauert, R.H.: Araldite as an embedding medium for electron microscopy. J. biophys. biochem. Cytol. 4, 191-194 (1958) Monneron, A.: Utilisation de la pronase en cytochimie ultrastructurale. J. Microscopic 5, 583-596 (1966) Monneron, A., Bernhard, W.: Action de certains enzymes sur des tissus inclus en ~pon. J. Microscopic 5, 697-714 (1966) Petit, A.: Contribution h l'6tude de l'6piphyse des Reptiles: le complexe ~piphysaire des Lacertiliens et l'6piphyse des Ophidiens. Etude embryologique, structurale, ultrastructurale; analyse qualitative et quantitative de la s&otonine dans des conditions normales et exp~rimentales. Th~se, Strasbourg (1976) Pevet, P.: The pineal gland of the mole (Talpa europaea L.). I. The fine structure of the pinealocytes. Cell Tiss. Res. 153, 277-292 (1974) Pevet, P.: 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 (1976) Pevet, P.: On the presence of different populations of pinealocytes in the mammalian pineal gland. J. Neural Transm. 40, 289-304 (1977) Pevet, P., Kappers, J. Ari~ns, Nevo, E.: The pineal gland of the mole-rat (Spalax ehrenbergi, Nehring). I. The fine structure of pinealocytes. Cell Tiss. Res. 174, 1-24 (1976) Pevet, P., Smith, A.R.: The pineal gland of the mole (Talpa europaea L.). II. Ultrastructural variations observed in the pinealocytes during different parts of the sexual cycle. J. Neural Transm. 36, 227-248 (1975) Quay, W.B., Renzoni, A., Eakin, R.M.: Pineal ultrastructure in Melopsittaeus undulatus with particular regard to cell types and functions. Riv. Biol. 61, 371-393 (1968) Reynolds, E.S.: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Roux, M., Richoux, J.P.: Etude ultrastructurale de l'6piphyse du L6rot (Eliomys quereinus L.) vivant dans des conditions normales et exp6rimentales. J. micr. Biol. Cell. 22, 33a-34a (1975) Roux, M., Richoux, J.P., Dussart, G.: Etude ultrastructurale de l'6piphyse du L6rot (Eliomys quercinus L.). Bull. Ass. Anat. (Nancy) 58, 1-12 (1974) Venable, J.M., Coggeshall, W.: Simplified lead stain for use in electron microscopy. J. Cell Biol. 25, 407-408 (1965) Vivien-Roels, B.: L'6piphyse des Ch61oniens. Etude embryologique, structurale, ultrastructurale; analyse qualitative et quantitative de la s6rotonine dans des conditions normales et exp6rimentales. Th6se, Strasbourg (1976)
Accepted May 17, 1977
Cell Tiss. Res. 182, 215-219 (1977)
9 by Springer-Verlag 1977
The Pineal Gland of the Mole (Talpa europaea L.) IV. Effect of Pronase on Material Present in Cisternae of the Granular Endoplasmic Reticulum of Pinealocytes P. Pevet Netherlands Institute for Brain Research, Amsterdam, The Netherlands
Summary. The effect of a proteolytic enzyme, pronase, on material present in cisternae of the granular endoplasmic reticulum of mole pinealocytes demonstrates their proteinaceous nature. Key words: Pinealocyte - Mole Proteinaceous material - Pronase.
Granular endoplasmic reticulum -
R6snm6. L'6tude des effets de l'action d'une enzyme prot6olytique, la pronase, sur les accumulations de mat6riel pr6sentes dans les citernes du r6ticulum endoplasmique granulaire des pin6alocytes de la Taupe, a permis de d6montrer leur nature prot6ique.
Introduction Accumulation of material in single cisternae of the granular endoplasmic reticulum (GER) or between the two layers of the nuclear envelope, has been observed in some pinealocytes of the dormouse (Roux et al., 1974; Roux and Richoux, 1975), the mole (Pevet, 1974, 1976; Pevet and Smith, 1975), the mole-rat (Pevet, 1976; Pevet et al., 1976) and some pineal parenchymal cells of the parakeet (type II, Quay et al., 1968). Observations reported earlier (see Pevet, 1976, 1977) led to the hypothesis that this material represents an exacerbation of a secretory process present in the pinealocytes of numerous mammals and non-mammals, the secretory process probably being involved in the synthesis of an antigonadotropic compound(s) (see details in Pevet, 1976, 1977). Similar accumulation of material has also been observed in several other cell types (see Fawcett, 1966) and was assumed to consist of protein (Fawcett, 1966; Cresti et al., 1974). In tissue embedded in epoxy resins, Monneron (1966) and Monneron and Bernhard (1966) have demonstrated that proteinaceous inclusions in pancreatic Send offprint requests to: Dr. P. Pevet, Netherlands Institute for Brain Research, IJdijk 28, Amsterdam-O, The Netherlands Acknowledgements: The author wishes to thank Prof. Dr. J. Ari~ns Kappers for his help in the present work and for revision of the manuscript, Miss M.T. Mud and Mr. P.S. Wolters for their skillful assistance and Mrs. M.M. Smidt for typing the manuscript
216
P. Pevet
cells o r in a d e n o h y p o p h y s i a l cells a r e d i g e s t e d b y p r o t e o l y t i c e n z y m e s s u c h as pronase and pepsin. In order to investigate the eventual proteinaceous nature o f t h e m a t e r i a l o b s e r v e d in t h e c i s t e r n a e o f t h e G E R o f s o m e p i n e a l o c y t e s , p r o t e o l y t i c e n z y m e s w e r e e m p l o y e d in t h e p r e s e n t study. T h e m o l e (Talpa europaea L.) w a s c h o s e n b e c a u s e t h e m a j o r i t y o f t h e m a t e r i a l a c c u m u l a t e d in t h e G E R o f this a n i m a l s h o w a p a r a c r y s t a l l i n e s t r u c t u r e (Pevet, 1974, 1976; P e v e t a n d S m i t h , 1975), w h i c h is r e a d i l y i d e n t i f i a b l e . P r o n a s e is a p o w e r f u l p r o t e o l y t i c e n z y m e w h i c h h y d r o l y s e s a l m o s t all p e p t i d e b o n d s in p r o t e i n s ( M o n n e r o n , 1966) a n d t h e r e f o r e a p p e a r e d to be a n a p p r o p r i a t e c h o i c e in t h e s e i n v e s t i g a t i o n s .
Material and Methods All four moles (3 females and 1 male) used in this study were captured in Deux-S~vres (France) in January, 1977. The animals were decapitated under ether anesthesia; the skull was opened and the region of the pineal flooded with cold fixative (2.5~ glutaraldehyde in 0.1 M phosphate buffer, pH7.25 at 4~ After its removal, the gland was placed in the same fixative at 4~ for 1 h. After washing in phosphate buffer for 12 h, the organs were dehydrated (without post-fixation) and embedded in araldite (Glauert and Glauert, 1958). Ultrathin sections were cut on a LKB ultramicrotome with glass knives and then collected on gold grids.
Enzymatic Extractions. The sections were oxidized for 30 min in a 10~ periodic acid solution, rinsed several times with distilled water and then floated for 30min, 1, 4 or 24h at 40~ on an enzymatic solution containing 0.5~o pronase (Koch-Light Laboratories Ltd, 45000 P.U.K./g) in distilled water, pH 7.4 (adjusted with NaOH 0.01 N). For controls, the last bath did not contain pronase. After enzymatic extraction, the sections were washed several times with distilled water and stained with uranyl acetate and lead citrate (Reynolds, 1963; Venable and GoggeshaU, 1965). Observations were made with a Philips 200 electron microscope.
Results A f t e r 30 a n d 60 m i n o f e n z y m a t i c t r e a t m e n t , t h e t y p i c a l p a r a c r y s t a l l i n e s t r u c t u r e o f t h e m a t e r i a l a c c u m u l a t e d in t h e G E R was l a r g e l y i n t a c t , i n d i c a t i n g t h a t t h e p r o n a s e h a d little effect at t h e s e t i m e p e r i o d s . H o w e v e r , in a few p i n e a l o c y t e s a v i s i b l e d i g e s t i o n h a d t a k e n place.
Fig. l a-e. Normal mole pinealocyte and pinealocytes after four hours treatment with pronase. a Control section. Accumulation of material (arrows) showing a typical paracrystalline structure between the two layers of the nuclear envelope, x 31,100. b Control section. Material (arrows) accumulated in the cisternae of the GER showing a typical paracrystalline structure, x 26,000. e Control section. Accumulation of material between the two layers of the nuclear envelope (arrow) without apparent paracrystalline organization, x 59,000. d Section treated with pronase. Two accumulations of material (*) located in cisternae of the GER are digested by pronase. The arrow points to remains of the paracrystalline structure, the hydrolysis of which was incomplete, x 28,900. e Section treated with pronase. Two digested accumulations of material (*). In the material located between the two layers of the nuclear envelope, hydrolysis of the paracrystalline structure is incomplete (arrow). mit Mitochondria, n nucleus, P pinealocyte, x 39,000 Fig. 2. Control section. A granular vesicle (arrow) present near the Golgi apparatus (g) is observed in a pinealocyte; n nucleus, x 40,000
Effect of Pronase on Mole Pinealocytes
217
218
P. Pevet
Practically all accumulations were digested after four hours of enzymatic treatment (Fig. 1). Only in a few rare cases were fragments of paracrystalline structure still observed (Fig. 1), the hydrolysis of which was evidently incomplete. After twenty four hours of treatment, all accumulations appeared to have been digested by pronase. However, the general cellular morphology was not well preserved. In some control sections, granular vesicles were observed (Fig. 2). Such vesicles were never seen in the enzyme-treated sections. Discussion
The cytochemical results obtained in this study prove that the material accumulated in the cisternae of the GER or between the two layers of the nuclear envelope of mole pinealocytes is proteinaceous in nature. This result justifies the term "accumulation o f proteinaceous material" earlier used for these structures (Pevet, 1976, 1977) and may point to their physiological importance (see Pevet, 1977). From the observations that paracrystalline structures remain intact after 30 and 60 min of enzymatic treatment but evidence of digestion is visible suggests that additional proteinaceous material is also present and possesses a greater sensitivity to pronase. In previous investigations (Pevet, 1974, 1976; Pevet and Smith, 1975), proteinaceous material has been observed which lacks a paracrystalline organization. It seems that the material digested by the pronase during the first hour corresponds to that having no paracrystalline structure as judged from the observation of structures with exclusively paracrystalline organization after 30 and 60 rain of enzymatic treatment. Nevertheless, this observation does not permit the conclusion that the different types of proteinaceous material observed in the mole are different in nature. Other studies will be necessary to solve this problem. We do believe that these different types might represent only different developmental stages of the same formation (see Fig. 21 in Pevet, 1974). Another point of interest is the nature of the matrix of granular vesicles originating from the Golgi apparatus. The fact that these vesicles were observed in control sections, but not in those subjected to pronase may prove that their proteinaceous matrix had been digested by pronase. This would agree with the results of Collin and Meiniel (1972), Vivien-Roels (1976) and Petit (1976) who, using the same technique, demonstrated the proteinaceous nature of the matrix of the granular vesicles in the pineal cells of lacertilians, magpies, tortoises and snakes ( N a t r i x n a t r i x ) . It can not be stated with certainty, however, that the matrix of the granular vesicles is actually digested by this enzyme. The granular vesicles are extremely scarce in normal mole pinealocytes (Pevet, 1974, 1976), so that positive identification of possibly digested granular vesicles in enzymatically treated sections is very difficult. References Collin, J.P., Meiniel, A.: L'organe pin6al du genre Lacerta (Reptile, lacertilien): action d'enzymes protrolytiques sur les grains denses (500-3400 A) des photorrcepteurs rudimentaires. C.R. Soc. Biol. (Paris) 166, 370-380 (1972)
Effect of Pronase on Mole Pinealocytes
219
Cresti, M., Pacini, E., Simoncioli, C.: Uncommon paracrystalline structures formed in the endoplasmic reticulum of the integumentary cells ofDiplotaxis erucoides ovules. J. Ultrastruct. Res. 49, 218-223 (1974) Fawcett, D.W.: An atlas of fine structure: The cell. Philadelphia and London: Saunders Co. 1966 Glauert, A.M., Glauert, R.H.: Araldite as an embedding medium for electron microscopy. J. biophys. biochem. Cytol. 4, 191-194 (1958) Monneron, A.: Utilisation de la pronase en cytochimie ultrastructurale. J. Microscopic 5, 583-596 (1966) Monneron, A., Bernhard, W.: Action de certains enzymes sur des tissus inclus en ~pon. J. Microscopic 5, 697-714 (1966) Petit, A.: Contribution h l'6tude de l'6piphyse des Reptiles: le complexe ~piphysaire des Lacertiliens et l'6piphyse des Ophidiens. Etude embryologique, structurale, ultrastructurale; analyse qualitative et quantitative de la s&otonine dans des conditions normales et exp~rimentales. Th~se, Strasbourg (1976) Pevet, P.: The pineal gland of the mole (Talpa europaea L.). I. The fine structure of the pinealocytes. Cell Tiss. Res. 153, 277-292 (1974) Pevet, P.: 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 (1976) Pevet, P.: On the presence of different populations of pinealocytes in the mammalian pineal gland. J. Neural Transm. 40, 289-304 (1977) Pevet, P., Kappers, J. Ari~ns, Nevo, E.: The pineal gland of the mole-rat (Spalax ehrenbergi, Nehring). I. The fine structure of pinealocytes. Cell Tiss. Res. 174, 1-24 (1976) Pevet, P., Smith, A.R.: The pineal gland of the mole (Talpa europaea L.). II. Ultrastructural variations observed in the pinealocytes during different parts of the sexual cycle. J. Neural Transm. 36, 227-248 (1975) Quay, W.B., Renzoni, A., Eakin, R.M.: Pineal ultrastructure in Melopsittaeus undulatus with particular regard to cell types and functions. Riv. Biol. 61, 371-393 (1968) Reynolds, E.S.: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Roux, M., Richoux, J.P.: Etude ultrastructurale de l'6piphyse du L6rot (Eliomys quereinus L.) vivant dans des conditions normales et exp6rimentales. J. micr. Biol. Cell. 22, 33a-34a (1975) Roux, M., Richoux, J.P., Dussart, G.: Etude ultrastructurale de l'6piphyse du L6rot (Eliomys quercinus L.). Bull. Ass. Anat. (Nancy) 58, 1-12 (1974) Venable, J.M., Coggeshall, W.: Simplified lead stain for use in electron microscopy. J. Cell Biol. 25, 407-408 (1965) Vivien-Roels, B.: L'6piphyse des Ch61oniens. Etude embryologique, structurale, ultrastructurale; analyse qualitative et quantitative de la s6rotonine dans des conditions normales et exp6rimentales. Th6se, Strasbourg (1976)
Accepted May 17, 1977
