Ultrastructural changes of the carp (Cyprinus carpio) hepatocyte ...

Biol. Cell (2006) 98, 457–463 (Printed in Great Britain)

doi:10.1042/BC20060006

Research article

Ultrastructural changes of the carp (Cyprinus carpio) hepatocyte nucleolus during seasonal acclimatization Marco Alvarez*, Claudia Quezada*, Alfredo Molina*, Manuel Krauskopf*, M. Ines Vera* and Marc Thiry†1 ´ ´ Bello, Santiago, Chile, and Millennium Institute for Fundamental and Applied *Departamento de Ciencias Biologicas, Universidad Andres ` Biology, Santiago, Chile, and †Laboratory of Cell Biology, Department of Life Sciences, Faculty of Sciences, University of Liege, ` Liege, Belgium

Background information. The eurythermal fish carp (Cyprinus carpio) adjusts to the seasonal changes in the temperature and photoperiod of its habitat through diverse cellular and molecular mechanisms. We have observed that ribosomal biogenesis is modulated during the acclimatization process and correlates with profound phenotypic changes, reflecting a seasonal-dependent ultrastructural appearance of the nucleolar components. Previous studies using classical techniques showed that in winter-adapted carp the nucleolus appears to be segregated. In the present work, we have reassessed the nucleolar ultrastructural organization of the carp in summer- and winter-adapted fish by using more specific cytochemical and immunocytological techniques. Results. The acetylation method provided evidence that the nucleolar organization is different between winter- and summer-adapted carp. In winter-adapted fish the fibrillar component appears as a unique mass surrounded by several granular caps, whereas in summer-adapted carp the fibrillar component forms few cordons surrounded by granular masses. The nucleolar structure and distribution of the condensed chromatin observed varies upon seasonal acclimatization. In winter the nucleolar chromatin is densely packed in masses that surround the nucleolus, whereas during summer it displays a rather looser organization formed by filaments that not only surround the nucleolus, but also go through the nucleolar body. Using the TdT (terminal deoxynucleotidyl transferase)– immunogold labelling technique, we detected condensed and decondensed nucleolar chromatin, and found some labelling of fibrillar components in both seasons. When liver tissue from summer-adapted carp was treated with AMD (actinomycin D), we observed that the rearrangement of the nucleolar components and condensed chromatin were similar to that found in winter-adapted fish, with differences in the distribution of the perinucleolar chromatin. Conclusions. The acetylation and TdT–immunogold labelling experiments indicated that the rearrangement of the nucleolar components of winter-adapted carp is very similar to the AMD-treated summer-adapted carp nucleolus, with the latter representing the repression of the ribosomal biogenesis that occurs during the cold season. Nevertheless, the distribution of the condensed perinucleolar chromatin in winter-adapted carp compared with AMD-treated cells suggests that the transcription of rRNA genes in winter-adapted fish is less strongly inhibited and does not lead to the classical segregation of the nucleolus of that described after AMD treatment. In addition, we have confirmed that carp hepatocyte nucleoli comprise only two main structural compartments: a fibrillar component and a granular component. Fibrillar centres were not observed.

1 To

whom correspondence should be addressed (email [email protected]). Key words: Cyprinus carpio (carp), nucleolus, ultrastructural changes, seasonal acclimatization. Abbreviations used: AMD, actinomycin D; TdT, terminal deoxynucleotidyl transferase.

Introduction The process of seasonal acclimatization observed in the teleost fish Cyprinus carpio (carp) sustains the physiological compensatory mechanisms that the organism has to survive the natural changes occurring

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M. Alvarez and others

in its habitat (Alvarez et al., 2004). The adaptive responses are associated with the seasonal-dependent cyclical regulation of transcriptional and translational events, and, in particular, with the transcription of ribosomal genes within the nucleolus. The structure of the nucleolus is largely dependent on the requirement of ribosomes by the cell. Ribosome formation is a highly dynamic and co-ordinated multistep process, which requires synthesis, processing and modification of pre-rRNA, its assembly with ribosomal proteins and transient interaction of numerous non-ribosomal factors with the pre-ribosomal particles (Tschochner and Hurt, 2003). The repression of the ribosomal genes during mitosis (Hernandez-Verdun et al., 2002) and after AMD (actinomycin D) treatment (Schofer et al., 1996) affects the structure and the integrity of the nucleolus, which results in changes in the relative abundance of several nuclear proteins and segregation of the nucleolar components (Andersen et al., 2002, 2005). In human meiotic oocytes, where the expression of the ribosomal genes is transitionally inactivated, the nucleolus also exhibits segregation of the nucleolar components (Hartung et al., 1979). In previous studies, we and others have demonstrated that upon winter acclimatization of carp, a physiological repression of the transcription of the ribosomal genes occurs, thus modulating ribosome biogenesis (Vera et al., 1993, 1997, 2000; Molina et al., 2002; Alvarez et al., 2003; Gracey et al., 2004). Previously, we have shown that during the cold season the carp nucleolus exhibited profound phenotypical changes (Sáez et al., 1984). To further our knowledge of the dynamic arrangement of the nucleolar components in summer- and winter-adapted carp hepatocytes, we assessed the detailed structure of this subnuclear compartment using a cytochemical and immunocytological experimental approach. We compared the cyto-architecture of the nucleolus, as well as the nucleolar distribution of the key constituents of the nucleolus, in hepatocytes of summer- and winter-adapted carp, and show that the hepatocytes from fish adapted to the cold season have nucleoli which are not classically segregated. Accordingly, we have reassessed the previous interpretations that were based on classical ultrastructural analyses (Sáez et al., 1984; Vera et al., 1993, 1997; Pinto et al., 2005). Our data suggest that the seasonal-dependent phenotype reprogramming induces a transition from a re-

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ticulated to a compact arrangement of the nucleolar components.

Results To visualize the different nucleolar components in the carp hepatocyte nucleolus, we used the acetylation method which allows an excellent distinction between the various nucleolar compartments (Wassef et al., 1979; Thiry et al., 1985). In addition, it offers the possibility to identify the condensed chromatin blocks situated around or inside the nucleolar body (Wassef et al., 1979; Thiry et al., 1985). The photomicrographs show that the nucleolus from summer-adapted carp (Figure 1A) appears less compact than the winter-adapted carp (Figure 1B). In both winter and summer, the carp hepatocyte nucleolus is composed of two main structural components: a fibrillar component and a granular component. These components are well separated in both types of nucleoli. The centrally-located fibrillar component of the nucleolus is surrounded by the granular component. However, the nucleolar organization is different in these two physiological conditions. In the winter-adapted carp, the fibrillar component appears as a unique mass surrounded by several granular caps (Figure 1B). In the summer-adapted carp, the fibrillar component forms few cordons, which are in a very close proximity to the surrounding granular masses (Figure 1A). In addition to the fibrillar component and the granular component, the nucleolus of carp hepatocytes contains condensed chromatin. Interestingly, the structure and distribution of the condensed chromatin varies according to the physiological conditions. In the winter-adapted carp, the condensed chromatin appears as densely contracted masses which are mainly located around the nucleolus (Figure 1B), and only a few small clusters are present in the fibrillar component. In the hepatocyte nucleolus of summeradapted carp, the condensed chromatin exhibited a somewhat looser structural organization. It is formed by filaments that are found not only around the nucleolus, but also inside the nucleolar body, and, in particular, in the fibrillar component and in the transition area between the fibrillar component and the granular component (Figure 1A). The acetylation technique favours the detection of condensed chromatin. In order to detect all (both condensed and decondensed) chromatin associated

Seasonal changes of the carp liver nucleolus

Research article

Figure 1 Ultrastructure of the nucleolus from winter- and summer-adapted carp Thin sections of hepatocytes were treated according to the acetylation method. (A) and (B) show nucleolar structures from summer- and winter-adapted carp respectively. The nucleolus is composed of the fibrillar component (F) and the granular component (G). Moreover, it contains a perinucleolar shell of condensed chromatin (C). Some intranucleolar clusters of condensed chromatin are also indicated (arrows). Scale bar, 0.2 µm.

with the nucleolus, we combined the acetylation method with the TdT (terminal deoxynucleotidyl transferase)–immunogold labelling procedure, which is a well-known method for detecting DNA in situ with high sensitivity and specificity (Thiry, 1992). We found, in addition to the intense labelling of the intranucleolar and perinucleolar condensed chromatin, some labelling of the fibrillar component of nucleolus under the two physiological conditions (Figures 2A and 2B). In contrast, the granular component appears to be totally devoid of gold particles (Figures 2A and 2B). The quantification of the distribution of the gold particles (Table 1) confirmed these observations. To confirm that the ultrastructural changes in the winter-adapted carp nucleolus was the phenotypic expression of the decrease in the transcription of the ribosomal genes, summer-adapted carp liver sections were treated with AMD to repress transcription and the structure of the hepatocyte nucleolus was ex-

amined (Figure 3). Treatment with AMD induced the formation of a compact fibrillar zone surrounded by several granular masses (Figure 3). It also resulted in the appearance of large clusters of condensed chromatin in preferential contact with the fibrillar region (compare Figure 3 with Figure 1A). This AMDinduced nucleolar segregation was comparable with that observed in the winter-adapted carp liver (see Figure 1B), but the distribution of the perinucleolar condensed chromatin was not exactly the same. In both cases condensed chromatin is present around the nucleolus, however, it is preferentially found in contact with the fibrillar component in AMD-treated cells.

Discussion Our results reveal that the nucleolus of carp hepatocytes comprises two main distinct components: a homogenous fibrillar zone and a granular zone. In the


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Figure 2 Localization of DNA within the nucleolar structures The in situ TdT–immunogold labelling procedure (Thiry, 1992) was applied to thin sections of liver from summer- (A) and winter-adapted (B) carp. Arrows indicate intranucleolar clusters of condensed chromatin. Labelling of the different nucleolar compartments is as described for Figure 1. Scale bar, 0.2 µm.

Table 1 Densities of gold particles in the fibrillar zone, the granular zone, the condensed chromatin and the resin of nucleoli from winter- and summer-adapted carp hepatocytes as revealed with the in situ TdT–immunogold labelling method The results are the means + − S.E.M. Sixteen and fourteen randomly chosen micrographs were analysed and the gold particles were counted (9242 and 6316 particles in total respectively). Student’s t test was used to compare the nucleolar components with the resin (*P < 0.1, **P < 0.01). Density of gold particles (particles/µm2 ) Fibrillar zone Granular zone Condensed chromatin Resin

Winter

Summer

6.01 + − 2.45* 2.13 + − 1.06 + 58.62** 247.95 −

4.49 + − 1.14* 1.13 + − 0.42 + 28.93** 149.02 −

1.65 + − 0.66

0.74 + − 0.15

nucleoli of summer- and winter-adapted carp, we did not observe the presence of fibrillar centres, which are typically found in the mammalian nucleus, even in the segregated nucleoli observed after treatment with AMD. Such specific nucleolus organization appears to be a characteristic not only in fish (Raikova,

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1976; Azevedo and Coimbra, 1980; Moyne and Collenot, 1982; Thiry and Poncin, 2005), but also for many other animals, including amphibians and insects (reviewed in Thiry and Goessens, 1996; Thiry and Lafontaine, 2005). Although we also found that the nucleolar organization changes during carp acclimatization were in agreement with previous studies (Sáez et al., 1984; Vera et al., 1993), the cytochemical and immunocytological approach provided new insights into the detailed morphological rearrangement. Indeed, during the cold season the nucleoli of hepatocytes do not appear to segregate as classically described (Sáez et al., 1984; Vera et al., 1993). In the segregated nucleoli, the perinucleolar-condensed chromatin disappears and only a few contacts persist between the condensed chromatin and the fibrillar component (Simard et al., 1974). As previously described by our laboratory (Sáez et al., 1984; Vera et al., 1993; Pinto et al., 2005), in the nucleus of winter-adapted carp hepatocyte, a layer of condensed chromatin around the nucleolus was always observed. In addition, in

Research article


Figure 3 Nucleolar components segregation in hepatocytes after treatment with AMD Thin sections of liver from summer-adapted carp were incubated with AMD (1 µg/ml) for 6 h then fixed for observation with electron microscopy. Labelling of the different nucleolar compartments is as described for Figure 1. Scale bar, 1.0 µm.

hepatocytes from summer-adapted carp, the nucleolar components are not highly intermingled, as previously reported (Sáez et al., 1984), but occupy distinct nucleolar areas. These discrepancies might likely reflect the difficulties in distinguishing the various nucleolar components using the classical methods used in the previous ultrastructural observations (Sáez et al., 1984). It has been shown that stimulation of resting cells is associated with changes in the ultrastructural aspects of their nucleolus. A typical example is the activation of resting human lymphocytes where the compact nucleolus in the resting cells is altered upon activation and becomes reticulated in the activated cells (Smetana and Busch, 1974). It is interesting to note that the structural alterations in the organization of the carp hepatocyte nucleolus during seasonal acclimatization are reminiscent of that observed upon activation of human lymphocytes (Smetana and Busch, 1974). As the transcription of the carp ribosomal genes and the expression of several proteins (i.e. ribosomal protein L41 and the protein kinase CK2β

subunit) involved in different steps of ribosomal biogenesis are down-regulated in winter (Vera et al., 1993, 1997, 2000, 2003; Molina et al., 2002), the ultrastructural aspect of the winter carp nucleus would be the consequences of these phenomena. Accordingly, the treatment of summer-adapted carp liver sections with AMD, which results in a repression of ribosomal gene transcription, leads to the appearance of structural features of the nucleolus comparable with that observed in the winter-adapted carp nucleolus. Nevertheless, the selective association of perinucleolar condensed chromatin with the fibrillar zone in AMD-treated cells seems to indicate that in the winter-adapted carp liver nucleolus the transcription of rRNA genes is less strongly inhibited than under AMD conditions.

Materials and methods Electron microscopy

Male carp (Cyprinus carpio) were captured and maintained under summer (20–22◦ C) and winter (8–10◦ C) temperatures (Pinto et al., 2005). Liver fragments from winter- and summer-adapted


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carp were sliced and immediately fixed for 2 h at room temperature in 0.1 M sodium phosphate (pH 7.4) containing 1% glutaraldehyde, 1% acrolein and 1% paraformaldehyde. After three washes in phosphate buffer, the samples were post-fixed in 1% osmium tetroxide, dehydrated with graded ethanol solutions and then processed for embedding in Epon/Araldite. Ultrathin sections of the samples were mounted on gold grids. The latter were incubated in pure pyridine for 10 s at room temperature, in pyridine/acetic anhydride (6:4) for 2 h at 45◦ C, in pyridine/ acetone for 10 s at room temperature, in acetone for 10 s and in ethanol for 10 s, and then dried. The ultrathin sections were stained with uranyl acetate and lead citrate before examination with a Jeol CX 100 transmission electron microscope at 60 kV. To determine the precise location of DNA in the nucleoli of fish cells, the in situ TdT–immunogold labelling procedure was used (Thiry, 1992). AMD treatment

Thin pieces of liver tissue from summer-adapted carps were incubated for 6 h at room temperature in Hanks balanced salt solution (Gibco) buffered with 10 mM Hepes (pH 7.4) (Sáez et al., 1982) and containing 1.0 µg/ml of AMD. Pieces of liver tissues incubated without AMD in parallel were used as controls. Once the incubation was completed, the samples were washed twice with PBS buffer at 4◦ C, fixed in 2.5% glutaraldehyde in 0.1 mM sodium cacodylate, and then embedded in Epon resin, which was allowed to polymerize for 48 h at 60◦ C.

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Received 5 January 2006/22 March 2006; accepted 27 March 2006 Published as Immediate Publication 27 March 2006, doi:10.1042/BC20060006


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