THE ANATOMICAL RECORD 230:319-324 (1991)
Influence of Age on Nuclear Bodies and Nuclear Volume in Pituicytes of the Rat Neurohypophysis M. LAFARGA, M.T. BERCIANO, J.M. PEREZ-FIGARES, M.A. ANDRES, AND E. MAQUIERA Department of Anatomy and Cell Biology, Faculty of Medicine, University of Cantabria, Santander (M.L., M.T.B., M.A.A., E.M.), and Department of Cell Biology, Faculty of Sciences, Malaga (J.M.P.-F.), Spain
ABSTRACT
This study has analyzed age-related changes in the nuclear organization of pituicytes of the rat. The cytological study of the cell nucleus and the quantitative analysis of nuclear bodies (NBs) were performed on ultrathin sections. Nuclear diameter, perimeter, and area were measured on semithin sections, and nuclear volume was estimated from these data. The nucleolus was mainly composed of a few large fibrillar centers with their associated dense fibrillar component, whereas the granular component tended to form large masses at the nucleolar periphery. The most frequent configuration of NBs was a globular inclusion composed of a fibrillar capsule with a core that contained a few electron-dense granules. Intranuclear glycogen was detected on rare occasions and only in old rats. The proportion of nuclear sections containing NBs increased significantly from 1.5% in 3-month-old rats to 8.6% in 18-month-old rats. A significant increase in the nuclear volume was detected in older rats with respect to the younger ones (157*69 vs. 98k43 pm3, meank53.D.). Our results suggest a n age-related activation of nuclear metabolism in pituicytes resulting in a nuclear expansion and a n increase in the frequency of appearance of NBs. This activation might be a reactive cellular event induced by the degenerative changes in neurosecretory nerve endings naturally occurring in older animals.
Several types of nuclear inclusions have been reported in glial cells. The “nuclear rodlets,” composed of a bundle of microfilaments, appear occasionally in ependymal cells, astrocytes, and oligodendrocytes both in normal (Field and Peat, 1971; Warchol, 1978) and in experimental and pathological conditions (Poppof and Stewart, 1968). A second variety of nuclear inclusions, the “nuclear bodies” (Bouteille e t al., 1967; DupuyCoin and Bouteille, 1972; Dupuy-Coin et al., 19721, are mainly composed of proteins and ribonucleoprotein particles. They sometimes appear in normal glial cells, particularly in older animals (Dahl, 1970; Field and Peat, 1971; Fiori, 1987), and under certain experimental and pathological conditions (Popoff and Stewart, 1968; Paula-Barbosa et al., 1980; Bertel and Gouranton, 1981). A third category of nuclear inclusion, the “coiled body,” seems to be a transitory nuclear structure in some immature glial cells (Lafarga et al., 1983). Moreover, the occurrence of intranuclear glycogen in glial cells has been reported in Muller’s cells “in vitro” (Amemiya, 1970), but not in the nuclei of any other glial cell lineages. It has been well established that the glial framework in the mammalian neural lobe is formed by pituicytes, a category of astroglial cells (Suess and Pliska, 1981) which establish dynamic interactions with the neurosecretory axons (Knowles and Vollrath, 1966; Sunde e t al., 1972; Tweedle and Hatton, 1980; Hatton, 1990). Although there is an extensive literature on the structure of pituicytes, both in normal and experimental 0 1991 WILEY-LISS, INC.
conditions (for review see Krsulovic and Bruckner, 1969; Wittkowski, 1986), the structural and functional organization of their cell nuclei has been neglected. The first objective of this study was to analyze the cytology and distribution of nuclear bodies (NBs) in the pituicytes of young and old rats. It has been reported in non-neural cells commonly containing NBs that cellular activation induces a n increase in the number of these inclusions as well a s progressive changes in their structural types (Radoux et al., 1984). Thus, the study of NBs in pituicytes may provide another structural feature for evaluating the response of these cells under different physiological and experimental conditions. The second objective was to study the agerelated changes in the nuclear volume of pituicytes, a parameter related to the transcription rate and the level of nucleocytoplasmic exchange (Herlan e t al., 1980; Brasch and Peters, 1985). The results are consistent with a n age-related activation of the nuclear metabolism in pituicytes, which induces nuclear expansion and a n increase in the frequency of appearance of NBs.
Received October 17, 1990; accepted December 5, 1990. Address reprint requests to Dr. Miguel Lafarga, Departamento de Anatomia y Biologia Celular, Facultad de Medicina, ciCardenal Herrera Oria sin, 39011 Santander, Spain.
320
M. LAFARGA ET AL.
ular NBs was rarely found in pituicytes from young The material studied consisted of neural lobe from 20 adult rats. All NBs were distributed randomly in the male Wistar rats of two age groups, 3 and 18 months nucleoplasm and they usually appeared with a clear old. Under anesthesia the animals were fixed by vas- nucleoplasmic halo around them (Figs. 3,4). Moreover, cular perfusion with 3%glutaraldehyde in 0.12M phos- no structural relations were observed between these phate buffer, pH 7.25. Next, the whole of the pituitary inclusions and the nucleolus or the nuclear envelope. Intranuclear glycogen was detected in pituicytes on gland was excised and the neurohypophysis was disrare occasions and only in old rats. Two patterns of sected and immersed in the same fixative overnight. The neurohypophysis was postfixed in 2% osmium intranuclear glycogen deposits were observed. The first tetroxide in phosphate buffer 0.12M, dehydrated in ac- consisted of a cluster of a few particles of the p-form, etone, and embedded in Araldite. The karyometric about 250 A in diameter (Fig. 5). These particles apanalysis of pituicytes was made on semithin sections, 1 peared in the interchromatin regions and preferenpm thick, stained with toluidine blue. The outlines of tially in the central portion of the nucleus. The second the cell nuclei were drawn with the aid of a camera pattern of glycogen accumulation consisted of a large, lucida attachment by using a x 100 oil objective. The centrally located glycogen body, up to 1.5 pm in diamdata were registered and statistically processed with a eter, which contained uniformly and closely packed @semiautomatic image analyzer (Videoplan, Kontron). particles (Fig. 6). This central agglomerate was surUltrathin sections were stained with 3% aqueous ura- rounded by a clear nucleoplasmic halo, the chromatin nyl acetate, followed by lead citrate, and examined in a being displaced to the nuclear periphery along the nuZeiss EM-10 electron microscope. The quantitative clear membrane. A reticular network of tenuous fibrils analysis of the number of NBs was performed by direct connected the periphery of the glycogen body with the ultrastructural observation of ultrathin sections. The marginal chromatin (Fig. 6). However, nuclear glycopercentage of pituicyte nuclear profiles containing NBs gen particles were not related to nuclear membranes. was estimated on randomly chosen nuclear sections. The cytoplasm of pituicytes containing intranuclear Smaller nuclear profiles corresponding to tangential or glycogen showed a normal ultrastructure, but dispolar sections of the cell nucleus were not included in played abundant lipid droplets (Fig. 6). Apart from the the count. Statistical tests used were analysis of vari- exceptional occurrence of intranuclear glycogen in oldr a t pituicytes, no apparent qualitative changes in the ance and Student’s t-test. cytology and organization of the cell nucleus were observed in old animals. MATERIALS AND METHODS
RESULTS UltrastructuralAnalysis of the Cell Nucleus
The ultrastructura1 study of the pituicytes dispIayed round to oval nuclei containing one or more nucleoli. A discontinuous band of condensed chromatin usually appeared aligned along the nuclear periphery, and some small clumps of chromatin were randomly distributed in the nuclear interior. Large masses of condensed chromatin were frequently associated with the nucleoli (Fig. 1).The nucleoli were generally eccentrically located and showed a peculiar organization of their components. In equatorial sections of the nucleolar body, one or a few large fibrillar centres were observed (Figs. 1, 2). These are round areas of low electron density composed of fibrillar material which are surrounded by the dense fibrillar component. The granular component usually appeared a s strands or clumps, which tended to be segregated a t the nucleolar periphery and separated from the dense fibrillar component by nucleolar interstices (Fig. 2). Nucleoli were partially surrounded by clumps of perinucleolar chromatin. A careful examination of the pituicyte cell nucleus allowed NBs to be found. Two subtypes of granular NBs were identified in the interchromatinic regions. The most frequent variety (about 90%) in both young adult and old rats was a globoid inclusion, 0.35 to 0.50 pm in diameter, composed of a dense fibrillar capsule of variable thickness that enclosed a loose fibrillar core containing a few dispersed electron-dense granules of about 125 A (Fig. 3a). As can be seen in Figure 3b, doublets with two granular cores were also observed. The second variety of granular NBs was a complex round inclusion, 0.5 to 0.7 pm in diameter, formed by a shell of fibrillar material surrounding a dense core of closely packed granules (Figs. 4a,b). This type of gran-
Morphometric Analysis
As can be seen in Table 1, the estimate of the proportion of nuclear profiles containing NBs showed a significant increase (P
Influence of Age on Nuclear Bodies and Nuclear Volume in Pituicytes of the Rat Neurohypophysis M. LAFARGA, M.T. BERCIANO, J.M. PEREZ-FIGARES, M.A. ANDRES, AND E. MAQUIERA Department of Anatomy and Cell Biology, Faculty of Medicine, University of Cantabria, Santander (M.L., M.T.B., M.A.A., E.M.), and Department of Cell Biology, Faculty of Sciences, Malaga (J.M.P.-F.), Spain
ABSTRACT
This study has analyzed age-related changes in the nuclear organization of pituicytes of the rat. The cytological study of the cell nucleus and the quantitative analysis of nuclear bodies (NBs) were performed on ultrathin sections. Nuclear diameter, perimeter, and area were measured on semithin sections, and nuclear volume was estimated from these data. The nucleolus was mainly composed of a few large fibrillar centers with their associated dense fibrillar component, whereas the granular component tended to form large masses at the nucleolar periphery. The most frequent configuration of NBs was a globular inclusion composed of a fibrillar capsule with a core that contained a few electron-dense granules. Intranuclear glycogen was detected on rare occasions and only in old rats. The proportion of nuclear sections containing NBs increased significantly from 1.5% in 3-month-old rats to 8.6% in 18-month-old rats. A significant increase in the nuclear volume was detected in older rats with respect to the younger ones (157*69 vs. 98k43 pm3, meank53.D.). Our results suggest a n age-related activation of nuclear metabolism in pituicytes resulting in a nuclear expansion and a n increase in the frequency of appearance of NBs. This activation might be a reactive cellular event induced by the degenerative changes in neurosecretory nerve endings naturally occurring in older animals.
Several types of nuclear inclusions have been reported in glial cells. The “nuclear rodlets,” composed of a bundle of microfilaments, appear occasionally in ependymal cells, astrocytes, and oligodendrocytes both in normal (Field and Peat, 1971; Warchol, 1978) and in experimental and pathological conditions (Poppof and Stewart, 1968). A second variety of nuclear inclusions, the “nuclear bodies” (Bouteille e t al., 1967; DupuyCoin and Bouteille, 1972; Dupuy-Coin et al., 19721, are mainly composed of proteins and ribonucleoprotein particles. They sometimes appear in normal glial cells, particularly in older animals (Dahl, 1970; Field and Peat, 1971; Fiori, 1987), and under certain experimental and pathological conditions (Popoff and Stewart, 1968; Paula-Barbosa et al., 1980; Bertel and Gouranton, 1981). A third category of nuclear inclusion, the “coiled body,” seems to be a transitory nuclear structure in some immature glial cells (Lafarga et al., 1983). Moreover, the occurrence of intranuclear glycogen in glial cells has been reported in Muller’s cells “in vitro” (Amemiya, 1970), but not in the nuclei of any other glial cell lineages. It has been well established that the glial framework in the mammalian neural lobe is formed by pituicytes, a category of astroglial cells (Suess and Pliska, 1981) which establish dynamic interactions with the neurosecretory axons (Knowles and Vollrath, 1966; Sunde e t al., 1972; Tweedle and Hatton, 1980; Hatton, 1990). Although there is an extensive literature on the structure of pituicytes, both in normal and experimental 0 1991 WILEY-LISS, INC.
conditions (for review see Krsulovic and Bruckner, 1969; Wittkowski, 1986), the structural and functional organization of their cell nuclei has been neglected. The first objective of this study was to analyze the cytology and distribution of nuclear bodies (NBs) in the pituicytes of young and old rats. It has been reported in non-neural cells commonly containing NBs that cellular activation induces a n increase in the number of these inclusions as well a s progressive changes in their structural types (Radoux et al., 1984). Thus, the study of NBs in pituicytes may provide another structural feature for evaluating the response of these cells under different physiological and experimental conditions. The second objective was to study the agerelated changes in the nuclear volume of pituicytes, a parameter related to the transcription rate and the level of nucleocytoplasmic exchange (Herlan e t al., 1980; Brasch and Peters, 1985). The results are consistent with a n age-related activation of the nuclear metabolism in pituicytes, which induces nuclear expansion and a n increase in the frequency of appearance of NBs.
Received October 17, 1990; accepted December 5, 1990. Address reprint requests to Dr. Miguel Lafarga, Departamento de Anatomia y Biologia Celular, Facultad de Medicina, ciCardenal Herrera Oria sin, 39011 Santander, Spain.
320
M. LAFARGA ET AL.
ular NBs was rarely found in pituicytes from young The material studied consisted of neural lobe from 20 adult rats. All NBs were distributed randomly in the male Wistar rats of two age groups, 3 and 18 months nucleoplasm and they usually appeared with a clear old. Under anesthesia the animals were fixed by vas- nucleoplasmic halo around them (Figs. 3,4). Moreover, cular perfusion with 3%glutaraldehyde in 0.12M phos- no structural relations were observed between these phate buffer, pH 7.25. Next, the whole of the pituitary inclusions and the nucleolus or the nuclear envelope. Intranuclear glycogen was detected in pituicytes on gland was excised and the neurohypophysis was disrare occasions and only in old rats. Two patterns of sected and immersed in the same fixative overnight. The neurohypophysis was postfixed in 2% osmium intranuclear glycogen deposits were observed. The first tetroxide in phosphate buffer 0.12M, dehydrated in ac- consisted of a cluster of a few particles of the p-form, etone, and embedded in Araldite. The karyometric about 250 A in diameter (Fig. 5). These particles apanalysis of pituicytes was made on semithin sections, 1 peared in the interchromatin regions and preferenpm thick, stained with toluidine blue. The outlines of tially in the central portion of the nucleus. The second the cell nuclei were drawn with the aid of a camera pattern of glycogen accumulation consisted of a large, lucida attachment by using a x 100 oil objective. The centrally located glycogen body, up to 1.5 pm in diamdata were registered and statistically processed with a eter, which contained uniformly and closely packed @semiautomatic image analyzer (Videoplan, Kontron). particles (Fig. 6). This central agglomerate was surUltrathin sections were stained with 3% aqueous ura- rounded by a clear nucleoplasmic halo, the chromatin nyl acetate, followed by lead citrate, and examined in a being displaced to the nuclear periphery along the nuZeiss EM-10 electron microscope. The quantitative clear membrane. A reticular network of tenuous fibrils analysis of the number of NBs was performed by direct connected the periphery of the glycogen body with the ultrastructural observation of ultrathin sections. The marginal chromatin (Fig. 6). However, nuclear glycopercentage of pituicyte nuclear profiles containing NBs gen particles were not related to nuclear membranes. was estimated on randomly chosen nuclear sections. The cytoplasm of pituicytes containing intranuclear Smaller nuclear profiles corresponding to tangential or glycogen showed a normal ultrastructure, but dispolar sections of the cell nucleus were not included in played abundant lipid droplets (Fig. 6). Apart from the the count. Statistical tests used were analysis of vari- exceptional occurrence of intranuclear glycogen in oldr a t pituicytes, no apparent qualitative changes in the ance and Student’s t-test. cytology and organization of the cell nucleus were observed in old animals. MATERIALS AND METHODS
RESULTS UltrastructuralAnalysis of the Cell Nucleus
The ultrastructura1 study of the pituicytes dispIayed round to oval nuclei containing one or more nucleoli. A discontinuous band of condensed chromatin usually appeared aligned along the nuclear periphery, and some small clumps of chromatin were randomly distributed in the nuclear interior. Large masses of condensed chromatin were frequently associated with the nucleoli (Fig. 1).The nucleoli were generally eccentrically located and showed a peculiar organization of their components. In equatorial sections of the nucleolar body, one or a few large fibrillar centres were observed (Figs. 1, 2). These are round areas of low electron density composed of fibrillar material which are surrounded by the dense fibrillar component. The granular component usually appeared a s strands or clumps, which tended to be segregated a t the nucleolar periphery and separated from the dense fibrillar component by nucleolar interstices (Fig. 2). Nucleoli were partially surrounded by clumps of perinucleolar chromatin. A careful examination of the pituicyte cell nucleus allowed NBs to be found. Two subtypes of granular NBs were identified in the interchromatinic regions. The most frequent variety (about 90%) in both young adult and old rats was a globoid inclusion, 0.35 to 0.50 pm in diameter, composed of a dense fibrillar capsule of variable thickness that enclosed a loose fibrillar core containing a few dispersed electron-dense granules of about 125 A (Fig. 3a). As can be seen in Figure 3b, doublets with two granular cores were also observed. The second variety of granular NBs was a complex round inclusion, 0.5 to 0.7 pm in diameter, formed by a shell of fibrillar material surrounding a dense core of closely packed granules (Figs. 4a,b). This type of gran-
Morphometric Analysis
As can be seen in Table 1, the estimate of the proportion of nuclear profiles containing NBs showed a significant increase (P
