MICROSCOPY RESEARCH AND TECHNIQUE 39:453–466 (1997) Biogenesis of Peroxisomes in Fetal Liver MARC ESPEEL,1 MARIANNE DEPRETER,1 ROBERTA NARDACCI,2 KATHARINA D’HERDE,1 INGRID KERCKAERT,1 STEFANIA STEFANINI,2 AND FRANK ROELS1 1Department 2Department of Anatomy, Embryology and Histology, Section of Anatomy and Embryology, University of Gent, B-9000 Gent, Belgium of Cellular and Developmental Biology, University of Rome ‘‘La Sapienza,’’ 00185 Rome, Italy KEY WORDS peroxisome; biogenesis; differentiation; liver; congenital disease; oocyte; inheritance ABSTRACT Peroxisomes are single membrane-limited cell organelles that are involved in numerous metabolic functions. Peroxisomes do not contain DNA; the matrix and membrane proteins are encoded by the nuclear genome. It is assumed that new peroxisomes are formed by division of existing organelles. The present article gives an overview of microscopic studies and recent unpublished results dealing with peroxisome biogenesis in mammalian fetal liver and presents data on peroxisomes in oocytes. Cytochemical (catalase and D-aminoacid oxidase activity) and immunocytochemical data in rat and human liver (antigens of catalase, the three peroxisomal b-oxidation enzymes, alanine: glyoxylate aminotransferase, peroxisomal membrane proteins with molecular weights of 42 and 70 kDa) indicate that during embryonic and fetal development the peroxisomal population undergoes a differentiation with respect to the composition of the matrix and to the size and number of the organelles. In the youngest stages, rare and small peroxisomes are present, into which the matrix components are imported in a sequential way. The import seems asynchronous in peroxisomes of the same hepatocyte. The size and number of the peroxisomes increase during liver development. In rat and human liver, no morphological or immunocytochemical evidence for an elaborate network of interconnected peroxisomes (‘‘reticulum’’) was found. Instead, peroxisomes presented as individual organelles, which occasionally show membrane extensions. The importance of the metabolic functions of peroxisomes in human liver is emphasized by the peroxisomal disorders. In the liver of affected fetuses, the microscopic features associated with the defect can already be recognized; i.e., either catalase containing peroxisomes are absent and catalase is localized in the cytoplasm (in fetuses affected with Zellweger syndrome or with infantile Refsum disease) or peroxisomes are present but they are abnormally enlarged (e.g., a fetus affected with acyl-CoA oxidase deficiency). In the quail ovary, numerous peroxisomes are observed in the oocyte and in the granulosa cells during follicle maturation, but not in the full-grown egg. Thus, the mechanism of peroxisome inheritance remains unresolved. Microsc. Res. Tech. 39:453–466, 1997. r 1997 Wiley-Liss, Inc. INTRODUCTION Peroxisomes are single membrane-bound organelles that are present in all eukaryotic cells. They belong to the family of microbodies, which includes peroxisomes, glycosomes, and glyoxysomes. The common feature of microbodies is that they are limited by a single membrane and that they share a fatty acid b-oxidation system, which is different from that in mitochondria. Peroxisomes occur in yeasts, protozoans, plant leaves, and animal cells. They do not contain DNA; their proteins are encoded by nuclear genes. The peroxisomal proteins are synthetized on free ribosomes; they are incorporated without posttranslational modifications into existing peroxisomes (except for 3-ketoacylCoA thiolase and sterol-carrier protein, which are synthesized as a precursor containing an aminoterminal presequence, and acyl-CoA oxidase, which is cleaved into two subunits; for reviews, see Borst, 1989; Lazarow and Fujiki, 1985; Van den Bosch et al., 1992). The first description of peroxisomes was given by Rhodin (1954) who designated spherical organelles in r 1997 WILEY-LISS, INC. the proximal tubules of mouse kidney as microbodies, and Rouiller and Bernhard (1956) described microbodies in the liver of the rat. By cell fractionation, De Duve and Baudhuin (1966) demonstrated that rat liver peroxisomes contain several hydrogen-peroxide-producing oxidases and catalase; the latter decomposes hydrogen peroxide in either a catalatic or a peroxidatic way. In mammalian peroxisomes, more than 40 distinct enzymes have been identified. The enzyme content of peroxisomes is tissue dependent; the conversion of a given substrate may be catalyzed by different enzymes Contract grant sponsor: Nationaal fonds voor Wetenschappelikj Onderzoek; Contract grant sponsor: Nationale Loterij; Contract grant number: 9.0027.88; Contract grant sponsor: Flemish Government, Verkennend Europees Onderzoek; Contract grant sponsor: European Biomed Concerted Action ‘‘Peroxisomal Leukodystrophy’’; Contract grant number: BMH4 CT96-1621 DGXII; Contract grant sponsor: University of Ghent, Bijzonder Ondersoeksfonds; Contract grant sponsor: Ministero dell’Università e della Ricerca Scientifica e Tecnologica. *Correspondence to: Marc Espeel, Department of Anatomy, Embryology and Histology, Section of Anatomy and Embryology, University of Gent, Godshuizenlaan 4, B-9000 Gent, Belgium. E-mail: email@example.com. Received 18 April 1995; Accepted in revised form 13 October 1995. 454 M. ESPEEL ET AL. in different species. Human liver peroxisomes are involved a.o. in the biosynthesis of plasmalogens, bile acids, cholesterol, and dolichol and in the degradation of very-long-chain fatty acids, phytanic acid, and the transamination of glyoxylate to glycin (for review, see Mannaerts and Van Veldhoven, 1993; Van den Bosch et al., 1992; Wanders et al., 1993). The size and shape of mammalian peroxisomes varies between cell types and between different species. In human and rat liver, the organelles have more or less a spherical shape; the matrix is finely granular (Sternlieb and Quintana, 1977; Wedel and Berger, 1975). Slender, vermiform peroxisomes cooccurring with spherical organelles are present in human duodenal epithelium (Roels et al., 1991a). Elongated peroxisomes are also common in the proximal tubules of human kidney (Roels and Goldfischer, 1979). In several cases, the peroxisomal matrix is divided into subcompartments. In rat liver peroxisomes, urate oxidase is present in the crystalline core (Völkl et al., 1988), and D-aminoacid oxidase is confined to an electron-lucent area of the matrix (Usuda et al., 1991a). In rat renal tubules, the peroxisomal matrix is divided into a peripheral electron-dense margin and an electronlucent central area. Some enzymes are predominantly present in the outer dense matrix (e.g., the peroxisomal b-oxidation enzymes), whereas others are exclusively localized in the central area (e.g., D-aminoacid oxidase) (Yokota et al., 1987). In normal human hepatic peroxisomes, there are no detectable subcompartments of the matrix. In pathological conditions, however, corelike inclusions have been reported: in patients with colorectal cancer (Denetto et al. (1991), in a specific subgroup of patients with primary hyperoxaluria type 1 (Danpure et al., 1993), in hepatic peroxisomes of a girl with a NALD-like syndrome (Roels et al., 1988), and in the hepatic and renal peroxisomes in a male neonate with a peroxisomal b-oxidation defect (Espeel et al., 1991). Rare hepatic peroxisomes with a marginal plate were present in a girl with a NALD-like syndrome (Roels et al., 1988). Peroxisomelike organelles with an electron-lucent central area have been reported in the liver of a patient with infantile Refsum’s disease (Roels et al., 1986, 1991b). Cytochemical investigations have demonstrated that, within the same tissue and even within the same cell, the organelles display heterogeneity regarding their enzyme activity: catalase activity differs between individual organelles in human liver (Roels, 1991; Roels and Cornelis, 1989) and staining heterogeneity for D-aminoacid oxidase activity has been shown in rat liver and kidney (Angermüller and Fahimi, 1988; Stefanini et al., 1985; also see Fig. 13 of the present article; for overviews and discussion, see Roels, 1991; Van den Munckhof (1996). In addition to the differences in the composition of the peroxisomal matrix among tissues, Usuda et al. (1991b) showed that the relative abundance of the membrane proteins differs among different rat organs. A remarkable feature of peroxisomes is that, in the liver of several mammalian species, their proliferation can be induced by administration of various chemical substances. The first observations of peroxisome proliferation induced by xenobiotics were made in rats (Hess et al., 1965). The molecular mechanism of the induction and the response mechanism have been intensively investigated. The proliferative response is species and gender dependent. Human liver peroxisomes do not respond to induction by these xenobiotics (for review, see Gibson and Lake, 1993), but proliferation in patients under several conditions has been described (De Craemer et al. 1991). Proliferation of the peroxisome population is also observed in regenerating rat liver after partial hepatectomy (Rigatuso et al., 1970). Peroxisome proliferation induced by drugs or by partial hepatectomy in rat liver has been used as a model for the study of mammalian peroxisome biogenesis (for review, see Fahimi et al., 1993; following section). The aim of the present article is to give an overview of microscopic studies on peroxisomes during mammalian liver organogenesis and in patients with peroxisomal disorder and to discuss the data with regard to peroxisome biogenesis. We also deal with the topic of peroxisome inheritance. PEROXISOME BIOGENESIS Two main processes are involved in the formation of a functional peroxisome: (1) the formation of the organelle as a membranous vesicle and (2) the incorporation of the matrix and the membrane proteins into this organelle. We use the term ‘‘biogenesis’’ to denote the process of organelle formation. The term ‘‘peroxisome assembly’’ is considered to be the result of processes (1) and (2). Organelle Formation For many years, the concept prevailed that peroxisomes were formed by budding from the endoplasmic reticulum (‘‘budding theory’’). In previous theories, peroxisomes were considered as precursors of mitochondria (Roullier and Bernhard, 1956) and of lysosomes (Bruni and Porter, 1965; for a discussion and overview, see de la Iglesia, 1969). The budding theory is based on early observations in rat liver that peroxisomes and endoplasmic reticulum often occur in close association. The existence of luminal continuities between both compartments was suggested in several electron micrographs prior to the use of cytochemical markers (Novikoff and Shin, 1964). Subsequent cytochemical and immunocytochemical studies, however, have demonstrated that both compartments are strictly separate entities (Shio and Lazarow, 1981; Yokota and Fahimi, 1980). Based on multiple evidence (see Lazarow and Fujiki, 1985), it is assumed that new peroxisomes originate from existing ones through division of a mother organelle. The ultrastructural basis of the formation of new peroxisomes has been studied in detail in the model of peroxisome proliferation by drug induction or by partial hepatectomy in rat liver (for review, see Fahimi et al., 1993). From these studies, the events that lead to the formation of a new organelle can be summarized as follows. The initial step is the formation of extensions of the peroxisomal membrane. They are attached to the ‘‘mother’’ peroxisome under the form of myelinlike figures that develop into looplike double-membrane structures. Upon import of the matrix proteins, the loops develop into small peroxisomes. They may remain 455 PEROXISOMES IN FETAL LIVER AND IN OOCYTES TABLE 1. Evolution of size and form-ellipse factor of peroxisomes in fetal human liver Fetal age (weeks) d-circle (µm) Mean uncorrected Maximum Form-ellipse factor Mean Minimum 8 10 12 14 14 16 16 16 16 17 0.316 0.621 0.329 0.605 0.376 0.633 0.382 0.687 0.381 0.664 0.383 0.679 0.394 0.663 0.342 0.536 0.456 0.888 0.508 0.779 0.782 0.433 0.728 0.249 0.906 0.613 0.914 0.704 0.887 0.658 0.805 0.388 0.854 0.408 0.854 0.579 0.862 0.630 0.918 0.767 briefly attached to the mother organelle but eventually separate to become ‘‘new’’ individual peroxisomes. The membrane extensions are strongly immunoreactive for the 70-kDa peroxisomal membrane protein and are considered as a reservoir ready for the import of the matrix proteins (Baumgart et al., 1989). The 70-kDa peroxisomal membrane protein is an ATP-binding protein that, through its homology with ABC-transporter proteins, is likely to be involved in the translocation of molecules across the peroxisomal membrane (Kamijo et al., 1990; Valle and Gärtner, 1993). In the same model, Lüers et al. (1990) demonstrated that the biosynthesis of the peroxisomal membrane proteins precedes that of the matrix proteins. Incorporation of the Matrix Proteins The peroxisomal matrix and membrane proteins are synthesized on free ribosomes. Two distinct targeting sequences that direct the matrix proteins to the peroxisomes have been identified so far in mammalian cells: a carboxy-terminal S-K-L (Ser-Lys-Leu) tripeptide is necessary and sufficient to direct a protein into the peroxisome (Gould et al., 1988). Limited variations of the SKL-tripeptide remain functional in peroxisomal targeting: serine may be replaced by alanine or cysteine, lysine may be substituted by arginine or histidine, but alternatives for the terminal leucine are not allowed. The minimal consensus sequence is thus S/A/C-K/R/ H-L; it is also called the S-K-L motif or PTS-1 (peroxisomal targeting signal). The S-K-L motif has been highly conserved during evolution (Gould et al., 1989). Swinkels et al. (1991) and Osumi et al. (1991) demonstrated that the aminoterminal cleavable extension of 3-ketoacylCoA thiolase contains a peroxisomal targeting sequence consisting of at least 11 amino acids, called PTS-2. The receptor for both the PTS-1 and PTS-2 targeting signal have been recently identified in man (Braverman et al., 1997; Dodt et al., 1995). The peroxisomal membrane proteins are targeted via different signals that have been identified in two proteins (McNew and Goodman, 1996; Wiemer et al., 1996). For reviews on peroxisomal protein sorting and import, we refer to Subramani (1993) and to Purdue and Lazarow (1994). Incorporation of newly synthesized peroxisomal proteins is taking place continuously in adult liver. Although biochemical studies have suggested that in adult liver peroxisomes as a whole have the same turnover rate as some of their proteins, there is little microscopic evidence for this. More likely, peroxisomes in healthy adult organs are relatively stable structures, but some of their components are continuously renewed, i.e., imported into existing structures. For a discussion on these subjects, see Roels (1991). Fig. 1. Visualization of peroxisomes in fetal human liver (menstrual age of 8 weeks) as tiny, dark granules after staining for catalase activity with diaminobenzidine. The organelles are abundant in the parenchymal cells. In addition, nucleated erythroblasts with black cytoplasm are seen. Semithin Epon section, phase contrast microscopy. 31,000. Bar, 10 µm. PEROXISOME BIOGENESIS AND PEROXISOMAL DISORDERS The role of peroxisomes in human metabolism is emphasized by the peroxisomal disorders, a group of rare inherited diseases in which one or more peroxisomal enzymes are deficient. Most often, it concerns very severe diseases with profound neurological impairment. 456 M. ESPEEL ET AL. Figs. 2–9. PEROXISOMES IN FETAL LIVER AND IN OOCYTES The peroxisomal disorders can be classified, on a biochemical basis, as three groups: the generalized peroxisomal disorders or peroxisome biogenesis disorders (e.g., Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease) with an impairment of most peroxisomal functions; the multiple peroxisomal enzyme deficiencies (e.g., rhizomelic chondodysplasia punctata); and the single peroxisomal enzyme deficiencies (e.g., X-linked adrenoleukodystrophy, primary hyperoxaluria type I, acyl-CoA oxidase deficiency) (Wanders et al., 1993). In group I, catalase containing peroxisomes are absent. In spite of the term ‘‘generalized’’ disorder, some peroxisomal functions may be unaffected. In patients with a peroxisomal disorder of group II and group III, hepatic peroxisomes are present. Except for X-linked adrenoleukodystrophy, they show diverse alterations (size, shape, number, appearance and electron density of the matrix after diaminobenzidine incubation). In addition, in all groups, other typical histopathological features can be present (for reviews, see Dimmick and Applegarth, 1993; Roels, 1991; Roels et al., 1991b, 1993b). The most severe clinical condition is the Zellweger syndrome or cerebro-hepato-renal syndrome; it is the prototype of the generalized peroxisomal disorder. Patients with Zellweger syndrome show craniofacial and skeletal malformations and severe neurologic impairments associated with neuronal migration defects; their life expectancy is less than 1 year (for review, see Fournier et al., 1994; Lazarow and Moser, 1995). Cytochemistry has shown that morphologically identifiable peroxisomes are absent in the liver and kidney of these patients (Goldfischer et al., 1973). At the time of this microscopic observation, the metabolic functions of peroxisomes were largely unknown; the relationship between the absence of peroxisomes and the biochemical profiles of these patients (accumulation of very-longchain fatty acids, phytanic acid, pipecolic acid, presence Fig. 2. Normal human fetal liver (14 weeks gestation). Staining intensity after diaminobenzidine incubation for catalase activity obviously differs among individual organelles. Usually larger organelles are less electron dense, indicating a lower catalase activity (Roels, 1991; Roels and Cornelis, 1989). 321,600. Bar, 0.5 µm. Fig. 3. Human fetal liver (18 weeks of gestation); cluster of peroxisomes with strong differences in catalase staining intensity. Many peroxisomes are seen in close association with cisternae of the endoplasmic reticulum (arrowheads). 326,400. Bar, 0.5 µm. Figs. 4, 5. Human fetal liver, 8 (Fig. 4) and 10 (Fig. 5) weeks of gestation. Tailed and elongated peroxisomes are sometimes seen; the image is reminiscent of peroxisome proliferation in adult regenerating rat liver (Yamamoto and Fahimi, 1987). Fig. 4: 356,000; bar, 0.25 µm. Fig. 5: 324,000; bar, 0.5 µm. Fig. 6. Human fetal liver (23 weeks of gestation). Peroxisomes with a somewhat angular shape exhibit different catalase staining. L, lipofuscin granule with heterogeneous contents. Numerous glycogen rosettes are seen. 325,000. Bar, 0.5 µm. Figs. 7–9. Human fetal liver (17 weeks of gestation). Peroxisomes are in close association with cisternae of granular (Fig. 7) and smooth endoplasmic reticulum. Although several peroxisomes are nearly in contact with each other, membrane continuities are not found. In Figure 8, adjacent peroxisomes have different concentrations of catalase reaction product, which argues against a continuous exchange of matrix components. In Figure 9, a cisterna of the endoplasmic reticulum is situated in between two peroxisomes (arrow). Fig. 7: 333,600; Fig. 8: 356,000; Fig. 9: 344,000. Bar, 0.5 µm. 457 of bile acid intermediates, deficiency of plasmalogens) became established later (Heymans et al., 1983; Schutgens et al., 1986). The absence of hepatic peroxisomes has also been demonstrated in patients with related, milder syndromes, e.g., infantile Refsum disease (Roels et al., 1986). The absence of peroxisomes was considered to result from a defect in organelle formation. Subsequent investigations have demonstrated that in cultured fibroblasts from patients with Zellweger syndrome ‘‘peroxisomal membrane ghosts’’ are present; these are enlarged and seemingly empty organelles, recognized as peroxisomes through the immunoreactivity of their membrane for peroxisomal membrane proteins. The membrane ghosts were considered to represent peroxisomes, which are unable to import the matrix proteins into the organelle (Santos et al., 1988a,b; Wiemer et al., 1989). The existence of ghosts in the liver of patients (Espeel et al., 1995a) is discussed below. Complementation studies with cultured skin fibroblasts from patients with generalized peroxisomal disorders have shown that these diseases are genetically heterogeneous. Eleven complementation groups in peroxisome biognesis disorders have been identified so far, suggesting that in man at least 11 different genes are involved in the formation of import competent organelles (Moser et al., 1995; Poulos et al., 1995; Shimozawa et al., 1993; Yajima et al., 1992). For some complementation groups, the involved gene and gene product have been identified: a 35-kDa peroxisomal membrane protein (PMP), called PAF-1 (peroxisome assembly factor1). Its precise role in peroxisome assembly is unknown; transfection of cultured skin fibroblasts from the patients with wild type PAF-1-cDNA restored peroxisome assembly and functioning. An experimental model for this complementation group is available: in a peroxisome deficient mutant line of Chinese hamster ovary cells (CHO-Z65), the defect is in the PAF-1 encoding gene (Shimozawa et al., 1992a; Tsukamoto, et al., 1991). Another complementation group is associated with the PXR1 gene, which encodes the PTS-1 receptor protein (Dodt et al., 1995). In addition, a defect in the gene encoding the 70-kDa PMP provokes a peroxisome biogenesis disorder (Gärtner et al., 1992). In addition to the CHO cell mutants (e.g. Shimozawa et al., 1992b; Thieringer and Raetz, 1993), various mutants of diverse yeast species are currently under intensive study to elucidate peroxisome biogenesis and assembly at the molecular level. The studies in yeast fall outside the scope of the present text (for a review, see Purdue and Lazarow, 1994). A unified nomenclature to designate the homologous genes in various organisms and their products (‘‘peroxins’’) responsible for peroxisome assembly has been introduced (Distel et al., 1996). PEROXISOMES IN FETAL LIVER Normal Fetal Human Liver1 (Overview) In a series of 15 fetal liver samples, with an in utero age of 8–23 weeks, Kerckaert (1990) described catalasecontaining peroxisomes in the parenchymal cells of all 1The observations refer to peroxisomes in the parenchymal cells. 458 M. ESPEEL ET AL. Fig. 10. Fetal liver of 7 weeks (menstrual age). Diaminobenzidine incubation revealed the cooccurrence of catalase-containing (arrowhead) and catalase-unreactive (star) peroxisomes. The arrow indicates an image of budding in a catalase-unreactive peroxisome. The inset shows three catalase-negative peroxisomes (star) present in a 6-week specimen (menstrual age). The matrix of the catalase-unreactive organelles is reticular in both samples. 327,600. Inset: 325,500. Bar, 0.2 µm. TABLE 2. Morphometry of peroxisomes in human liver Normal liver Fetal Postnatal 6 weeks1 7 weeks1 17 weeks2 20 weeks2 6 weeks2 4 months2 Adult3 Number measured d-circle (µm) Mean of profiles S.E.M. Mean corrected5 Maximum measured Volume density (%) Numerical density (µm23) Surface density (µm21) Form ellipse ‘‘Empty’’-looking peroxisomes in generalized peroxisomal disorders4 39 87 100 104 117 135 951 24 46 0.334 0.008 0.401 0.455 0.26 0.107 0.044 0.881 0.348 0.007 0.415 0.474 0.67 0.244 0.108 0.900 0.523 0.012 0.639 0.779 n.d.6 n.d. n.d. 0.918 0.512 0.010 0.620 0.742 n.d. n.d. n.d. 0.841 0.455 0.013 0.555 0.848 0.71 0.110 0.085 0.808 0.518 0.012 0.640 1.027 1.18 0.128 0.131 0.884 0.525 0.017 0.643 0.940 1.05 0.100 0.110 0.257 0.013 0.306 0.36 n.d. n.d. n.d. n.d. 0.237 0.009 0.280 0.41 n.d. n.d. n.d. n.d. 1Espeel et al. (1993a). et al. (1991b). samples; De Craemer et al. (1993). 4Two patients; Espeel et al. (1995a). 5Corrected for sectioning effect according to formula 9 from Abe et al. (1983). 6Not determined. 2Roels 3Seven samples. The size of the organelles (see Table 1) and their electron density after staining for catalase activity showed an increase during development; it reached a plateau value at the 17th week. Kerckaert (1990) also noticed the presence of irregularly shaped organelles, organelles with membrane extensions (‘‘tailed’’ peroxi- somes), clusters of peroxisomes, and a heterogeneity of staining for catalase activity between individual organelles. In the clusters of adjacent peroxisomes, cisternae of endoplasmic reticulum were present in between the organelles (Figs. 1–9). The presence of elongated peroxisomes was reflected in the low minimal value of the PEROXISOMES IN FETAL LIVER AND IN OOCYTES Fig. 11. Liver (18th week menstrual age) from a fetus affected with Zellweger syndrome. Immunostaining for catalase protein results in a diffuse reaction in the cytoplasm of the parenchymal cells; in many parenchymal cells, catalase immunoreactivity is also present in the nuclei. form-ellipse factor (i.e., the ratio between the long and the short axis of the object; see Table 1). Roels (1991) reported that in human fetal liver parenchyma images of budding (dividing) peroxisomes are rarely observed in these rapidly proliferating cells and that, in the clusters of peroxisomes, adjacent organelles display different amounts of diaminobenzidine reaction product. He concluded that if new organelles are formed by division of a mother organelle, the process of fission must proceed rapidly and that the separated mother and daughter organelles must have acquired their own individual enzyme content soon after the division. In a 6-week fetus (menstrual age), catalase-containing peroxisomes were not found after diaminobenzidine (DAB) cytochemistry; instead, small bodies with a reticular matrix and probably corresponding to a primitive stage of peroxisome development were present (Roels, 1991) (inset of Figure 10). In a 7-week specimen (menstrual age), similar catalase-negative organelles were present. They cooccurred in the same cell with catalase-positive organelles, the latter representing one-third of the population (Fig. 459 Fig. 12. Catalase immunostaining in infant control liver shows a granular reaction pattern, reflecting a peroxisomal localization of catalase; the nuclei are unreactive. 3750. Bar, 10 µm in Figs. 11, 12. Fig. 13. D-aminoacid oxidase cytochemistry in the liver of 15-day old fetal rat. Peroxisomes of different staining degrees are recognizable (arrowheads) in the hepatocyte cytoplasm; the star marks an unreactive peroxisome. 335,100. Bar, 0.2 µm. 460 M. ESPEEL ET AL. Fig. 14. Small peroxisome with diaminobenzidine reactive matrix in a presumptive hepatocyte of rat embryonic liver at 11.5 days postcoitum. Organelles are very rare at this stage. 372,000. Bar, 0.2 µm. Fig. 15. Small peroxisome with immunoreactive matrix for acylCoA oxidase in a presumptive hepatocyte of rat embryonic liver at 12.5 days postcoitum. 372,000. Bar, 0.20 µm. 10). In these young stages, the peroxisomes were distinctly smaller than in older fetuses and in postnatal liver (see Table 2). There were no significant size differences between the catalase-containing and the catalase-negative organelles. The catalase-positive organelles showed an extreme staining heterogeneity for catalase activity. This activity was considered, with the presence of the catalase-negative organelles, to reflect the onset of catalase import into the peroxisomes. In addition, we have suggested that in human embryonic liver catalase is imported in an asynchronous way (see Fig. 10) into individual, preexisting organelles (Espeel et al., 1993a). Only later during development do the three peroxisomal b-oxidation enzymes become detectable by immunocytochemistry: acyl-CoA oxidase and bifunctional enzyme in the 10th week and 3-ketoacylCoA thiolase in the 9th week (menstrual age). The immunoreactive signal for thiolase in simultaneously treated sections of the different stages showed a distinct increase of staining intensity with fetal age (Espeel et al., 1990). An increase of the intensity of catalase staining during development was also noticed (Kerckaert, 1990). Both observations indicate a progressive and continuous import of the peroxisomal matrix proteins during development. Our studies could not confirm the observation by de la Iglesia (1969) that in human fetal liver in the 6th week of gestation a nucleoid is present in the peroxisomal matrix. sent in the unprocessed form (Wanders et al., 1995) and that bifunctional enzyme is degraded (Tager et al., 1985; Wanders et al., 1995). In the liver of a 16-week fetus, in which previous microscopic and biochemical examinations of the chorionic villi demonstrated that it was affected with Zellweger syndrome (Roels et al., 1993a), catalase was immunolocalized in the cytoplasm of the parenchymal cells. Immunoreactivity for catalase was also present in at least a part of the nuclei (Fig. 11). This type of catalase immunolocalization is typically found in postnatal liver of patients with a generalized peroxisomal disorder but not in pre- or postnatal control liver (Fig. 12). Peroxisomes were also absent in the liver of a fetus with infantile Refsum disease (Poll-The et al., 1987). Enlarged peroxisomes have been reported in a fetus affected with acyl-CoA oxidase deficiency (De Craemer et al., 1991) and in a fetus with rhizomelic chondrodysplasia punctata (Roels et al., 1991b). Thus, the typical feature of enlarged peroxisomes in the postnatal liver associated with these syndromes (De Craemer et al., 1991; Heymans et al., 1986; Hughes et al., 1992; Poll-The et al., 1988) is already observed in the fetal liver. For a discussion on enlarged hepatic peroxisomes in peroxisomal disorder patients, see Roels (1991). Otherwise, in the liver of a stillborn fetus with X-linked recessive chondrodysplasia punctata (peroxisomal functions are normal with this disease; the fetus was delivered in the 26th postmenstrual week), no alterations of the peroxisomes were found (Van Maldergem et al., 1991). Fetal Liver Affected by Peroxisomal Disorders In the group I peroxisomal disorders (e.g., Zellweger syndrome), the peroxisomal matrix proteins are not imported into peroxisomes, and they remain localized in the cytoplasm. Despite this mislocalization, several enzymes remain stable and active (e.g., catalase, alanine/glyoxylate aminotransferase; Roels, 1991; Wanders et al., 1984, 1987). Immunoblotting studies have shown that in Zellweger liver the b-oxidation enzymes acyl-CoA oxidase and 3-keto-acylCoA thiolase are pre- Fetal Rat Liver (Overview) Without the use of cytochemical markers, Tsukada et al. (1968) described microbodylike organelles in fetal rat liver from 15 days onward (the youngest stage examined) and reported an evident increase of their number between the 15th and 18th days. Activity measurements showed that catalase, urate oxidase, and D-amino acid oxidase became detectable sequentially. Stefanini et al. (1985) found that catalase- PEROXISOMES IN FETAL LIVER AND IN OOCYTES Fig. 16. Granulosa cell of a primordial (prelampbrush) quail follicle after diaminobenzidine incubation for catalase activity. Five peroxisomes are recognized due to the precipitated diaminobenzidine; their membranes are clearly visible. Cytoplasm also contains mitochondria, rough endoplasmic reticulum, and free ribosomes. BM, basement membrane of the granulosal epithelium. 337,500. Bar, 0.5 µm. Fig. 17. Primordial follicle (prelampbrush stage) after diaminobenzidine incubation for catalase activity. G, monolayered granulosa with groups of rounded peroxisomes; O, oocyte with wormlike peroxisomes 461 (arrow); N, granulosal nuclei. Phase contrast image of semithin (2 µm) Epon section. 31,750. Bar, 10 µm. Fig. 18. Oocytal peroxisome with a tubular form in primordial follicle after diaminobenzidine incubation for catalase; membrane is clearly visible. A cisterna of smooth endoplasmic reticulum is in close proximity to the peroxisome. 360,000. Bar, 250 nm. Fig. 19. Catalase immunoreactivity in a prelampbrush oocyte; the gold particles are concentrated in a wormlike structure (cf. Fig. 19); the mitochondrial (M) profiles are devoid of gold particles. 336,000. Bar, 0.5 µm. 462 M. ESPEEL ET AL. containing organelles were present at the 14th day of intrauterine life and that D-amino acid oxidase activity was detectable from the 16th day. The import of the latter enzyme does not occur simultaneously in all peroxisomes because organelles with strong and weak reaction products coexist in the same cell with unreactive organelles (Fig. 13). Recent Studies in Human and Rat Liver Collaborative studies by our group and Stefanini’s group in young embryonic rat liver (10.5–15.5 days postcoitum) have provided new data on the differentiation of the peroxisome population during liver development. Already at 10.5 days, peroxisomes were visualized by their catalase activity in presumptive hepatocytes; they were very rare and small. At 11.5 days, catalase protein and the 43-kDa membrane protein were detectable in the peroxisomal matrix and membrane, respectively, and at 12.5 days immunoreactivity for the three peroxisomal b-oxidation enzymes was demonstrated in the matrix (Figs. 14, 15). In none of these stages was a nucleoid found; it was seen at 18 days. The size of the organelles increased from 13.5 days onward and at 15.5 days (d-circle 5 0.27 µm), it was almost twofold the value of the initial stages (10.5–12.5 days; d-circle 5 0.16 µm) but only two-thirds of that in adult rat liver. These are the youngest stages examined so far in rat liver. In human embryonic liver staged at 38 days postfertilization, peroxisomes contained immunoreactive catalase, AGT, acyl-CoA oxidase, 3-ketoacylCoA thiolase, and the peroxisomal membrane immunoreacted for the 43- and 70-kDa membrane proteins (Depreter et al., 1997). HEPATIC PEROXISOMES IN PEROXISOMAL DISORDER PATIENTS IN RELATION TO PEROXISOME BIOGENESIS In the liver of patients with a generalized peroxisomal disorder, rare and empty-looking organelles, identified as abnormal peroxisomes by the reactivity of their membrane for a 43-kDa peroxisomal membrane protein, were demonstrated. Two different types were identified, and they are considered to be the homologue of the ‘‘peroxisomal membrane ghosts’’ in the fibroblasts of Zellweger patients, i.e., organelles uncapable to properly import their matrix components (Espeel et al., 1995a). The size of these organelles is obviously smaller than that of the peroxisomes in postnatal control liver (Table 2). In other patients, a mosaic distribution of parenchymal cells with and without peroxisomes was found (Espeel et al., 1995b; Giros et al., 1995; Mandel et al., 1994; Roels et al., 1996a). Several hypotheses have been proposed to explain the mosaic distribution, for example, that the microenvironment (extracellular matrix) influences the differentiation of the peroxisome population. This hypothesis was inferred from the facts that (1) hepatocyte-specific features are determined by tissue interactions early during development (e.g. Caron, 1990; Cascio and Zaret, 1991) and (2) the number, size, shape, and catalase activity of peroxisomes in cultured hepatocytes depend on the culture conditions (Furukawa et al., 1988; Mitaka et al., 1993; Roels et al., 1996a). Thus far, studies of early embryonic rat liver have not confirmed a relationship between peroxisome maturation and the presence of extracellular matrix proteins laminin and collagen IV (Depreter et al., 1997). Another explanation for the mosaic distribution is derived from the observation that new peroxisomes originate by division of existing organelles. This notion implies that each eukaryotic cell should contain at least one peroxisome throughout its life. At the moment of mitotic division, the peroxisomes should be present in a number that is sufficient to guarantee that the organelles segregate to both daughter cells. If a daughter cell without peroxisomes were to originate, then all of its offspring should remain peroxisome deficient. Warren and Wickner (1996) distinguished a stochastic and an ordered inheritance of cell organelles. Recent studies have shown that in different cell types peroxisomes are associated with microtubules and that the organelles are capable of making directional movements along the microtubules (Rapp et al., 1996; Schrader et al., 1996; Wiemer et al., 1997). According to Wiemer et al., peroxisomes in CV1 cells (green monkey kidney cells) distribute themselves in a stochastic rather than an ordered way to the daughter cells at the time of mitosis, the majority of peroxisomes not being associated with microtubules of the mitotic spindle. PEROXISOMES IN OOCYTES In line with the concept of fission of existing peroxisomes to form new organelles, peroxisomes must be present in germ cells to be passed to the next generation. Goldfischer (1987) assumed, as it is the case for mitochondria, that peroxisomes are delivered to the zygote by the oocyte. Therefore, the identification of peroxisomes in oocytes is an intriguing question. We have examined by catalase cytochemistry and immunocytochemistry the presence of peroxisomes in follicles from the Japanese quail (Coturnix coturnix japonica) (Roels et al., 1996b). Oocyte Numerous diaminobenzidine reactive peroxisomes with a tubular shape are present in the oocytes during the small previtellogenic stages (follicle diameter 5 50– 500 µm; Fig. 17). The tubular organelles often show a constricted middle part and they are frequently lie in the vicinity of smooth endoplasmic reticulum cisternae (Fig. 18). After immunostaining against catalase, the label is concentrated over organelles with a similar size and shape as the DAB-reactive peroxisomes (Fig. 19). In the larger previtellogenic follicles (lampbrush stage II: 0.5–1.5 mm), the organelles are smaller and much fewer; their shape is spherical. In all vitellogenic stages (postlampbrush oocytes: 1.5–19 mm) examined so far, DAB-reactive peroxisomes were no longer observed. Granulosa Cells The granulosa cell layer in previtellogenic stages contains numerous and large round-to-ovoid peroxisomes; they sometimes have an angular outline and they occur in groups (Fig. 16). In vitellogenic follicles with diameters of 1.5–10 mm, peroxisomes are present, but their size (100–200 nm) is smaller than that in the previtellogenic stages (300–500 nm). Remarkably, in the three largest preovulatory follicles, i.e., yellow PEROXISOMES IN FETAL LIVER AND IN OOCYTES follicles with diameters of 10–19 mm, DAB-containing peroxisomes are no longer found. The observations are interpreted as follows. The image of elongated peroxisomes connected with a DABunreactive stalk in the small previtellogenic follicles represents morphological evidence for the formation of new organelles (cf. Baumgart et al., 1989). At this stage of oocyte development, new peroxisomes must be formed (biogenesis) at a high rate to match the large volume expansion of the oocyte. Indeed, peroxisomes are still detected in lampbrush II stage. The microscopic disappearance of peroxisomes in the vitellogenic oocytes is difficult to interpret. Volume expansion is enormous (a factor of 2,000 for growth from 1.5 to 19 mm oocyte) and could easily result in a ‘‘dilution effect.’’ A second explanation could be a stop in catalase synthesis during the last weeks of oocyte maturation: the normal enzyme turnover will result in organelles devoid of catalase. The absence of catalase-containing peroxisomes in the granulosa cells of mature follicles does not result from a volume expansion of the cells. It appears that these cells, which in birds die after ovulation, do not sustain a differentiated peroxisome population until the end of their life span. CONCLUSIONS, HYPOTHESES, AND FUTURE PROSPECTS The observations in fetal human and rat liver indicate that even in the youngest stages examined peroxisomes are detectable; they are very rare and small. During development, the matrix seems to obtain its complete composition in a stepwise way: e.g., in rat liver catalase (antigen and activity) is detectable prior to the three b-oxidation enzymes and the nucleoid in the rat liver peroxisomes appears still later. This finding suggests a sequential import, which seems to be asynchronous for individual organelles, given the interperoxisomal staining heterogeneity. The sequential and asynchronous import reflects a differentiation process (‘‘maturation’’) of individual peroxisomes, which is also associated with an increase of the size of the organelles. The factors governing this differentiation are unknown, and it is an open question as to whether these factors are specific for a given tissue. A parallel study of the peroxisome population of, e.g., the primitive gut endoderm outside the liver bud or of neuroepithelial cells could be relevant to this question. According to the observations by Depreter et al. (1997), there is no direct role for laminin or collagen IV in the differentiation of peroxisomes. With respect to the question of organelle inheritance, it is sufficient that peroxisomes are present in the ‘‘immature’’ form, provided their number is high enough to ensure segregation to the daughter cells. The necessity for a sufficient number of organelles implies that these immature organelles should be capable to divide. The empty-looking membrane ghosts in the postnatal liver of patients with a generalized peroxisomal disorder (into which import and incorporation of functional proteins have failed) could be considered as homologous to the immature peroxisomes, and they both share the feature of a small size. In view of the suggested differentiation process, the observations on peroxisomes during kidney embryonic development should be mentioned: whereas, in murine (Pipan and Psenicnik, 1975) and human fetuses (Bri- 463 ère, 1986), DAB-reactive peroxisomes are found as early as the 14th day and the 10th week of gestation, respectively, in rat proximal tubules the appearance of catalase-containing peroxisomes occurs as an abrupt phenomenon at the end of the prenatal period; in the preceding stages catalase-devoid organelles could not be detected (Stefanini et al., 1994). The dramatic increases of peroxisomal number and enzymatic content, found near birth in mouse and rat kidney proximal tubules, suggest a specific role for these organelles during the maturation of this part of the nephron (Goeckermann and Vigil, 1975; Pipan and Psenicnik, 1975; Stefanini et al., 1995). The existence of the membranous compartment (immature peroxisomes) in rat kidney at younger stages can be usefully investigated by immunocytochemistry for the peroxisomal membrane proteins. The cytochemical demonstration of peroxisomes in oocytes is a recently explored domain. During growth of the quail oocyte, peroxisomes apparently proliferate. In mature eggs, however, they can no longer be detected by catalase cytochemistry, which may be a result of the sensitivity of our methods or the matrix of the peroxisomes transiently may not contain catalase. The question remains open as to how peroxisomes are inherited by the zygote. ACKNOWLEDGMENTS We thank Dr. E. Jauniaux (Université Libre de Bruxelles, Belgium), Dr. N. Brière (Université de Sherbrooke, Québec, Canada), and Prof. P. Thorogood and Dr. C.T.J. Chan (Institute of Child Health, MRC Human Embryonic Tissue Bank, London, UK) for providing the fetal human liver samples. 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