Differentiation of liver peroxisomes in the foetal and newborn rat. Cytochemistry of catalase and D-aminoacid oxidase

0 Department of Biology, II University of Rome , Rome , Italy 1 A. Mow , 00185 Rome , Italy 2 Department of Cellular and Developmental Biology, I University of Rome , Piazzale Organules containing cytochemically detectable amounts of catalase and D-aminoacid oxidase activities are observed between the 14th and 21st day of development in the parenchymal cells of the foetal rat liver and in the liver of newborn rats. As early as 14 to 15 days, a limited number of small microperoxisomes, scattered in the cytoplasm of very few hepatocytes, can be found. These are roundish shaped, have a granulous matrix and contain very low, hardly detectable levels of the above mentioned enzymes. In later development both the size and the enzymatic content of the organules gradually increase, approaching adult levels at the end of foetal development. Starting from the 18th to 19th day of intrauterine life nucleoids can be seen in many peroxisomes. The morphological and biochemical maturation from microperoxisomes to peroxisomes is accompanied by a gradual increase in the number of stainable organules, both per individual cell and per tissue area. - different tissues. Following the first description of peroxisomes in rat hepatocytes (Rouiller & Bernhard, 1956), the electron microscopic features and the enzymatic complement of these organules have been reported in detail in a number of animal species and While the peroxisomal population has been extensively studied in the adult tissues, limited information is available concerning the embryogenesis of these organules. Data on peroxisome development have been obtained on rat (Tsukada, Mochizuki & Konishi, 1968), mouse (Essner, 1969) and chick (Essner, 1970) liver, on guinea pig adrenocortical cells (Black & Bogart, 1973) and on mouse 'microbodies' starting from 15 days of intrauterine life and reported the increases in the total number of organules and in the number of nucleoid-containing peroxisomes during pre- and postnatal development; in this study the morphological and morphometrical results were correlated with the biochemical data on catalase, D-aminoacid oxidase (D-AAO) and urate oxidase. At present, however, cytochemical studies on peroxisomes in foetal rat hepatocytes are still lacking, although specific and sensitive methods, such as the 3,3'diaminobenzidine (DAB) technique for catalase (Graham & Karnovsky, 1966; Fahimi, 1969; Novikoff & Goldfisher, 1969) and the cerium method for peroxisomal oxidases (Briggs, Drath, Karnovsky & Karnovsky, 1975; Veenhuis & Wendelaar Bonga, 1977) have been introduced and extensively applied to the study of peroxisomes in various tissues, mostly adult. It seemed worthwhile, therefore, to study the pre- and perinatal development of rat liver peroxisomes through their responsiveness to catalase and D-AAO cytochemical reactions. The application of these methods could reveal nascent organules more specifically and at earlier times with respect to morphological or biochemical techniques, thus allowing, for the first time, a cytochemical characterization of the peroxisome population in the developing hepatocyte. The appearance, morphology, distribution and size of catalase and D-AAO containing organules have been studied as from 13th day of intrauterine life to birth. For each developmental stage the number of peroxisomes per thin section of cell was also determined; values were examined by keeping in mind data reported by Greengard, Federman & Knox (1972) and by Herzfeld, Federman & Greengard (1973) on the volumetric variations of foetal hepatocytes and on the differentiation of other organules. MATERIAL AND METHODS Albino Wistar rats were commercially obtained and fed a standard laboratory diet and tap water 'ad libitum'. The females were placed with males overnight and examined the following morning for presence of sperm in the vaginal smear. The day of sperm observation was considered day 1 of pregnancy. Foetal age was further confirmed using the tables of Stotsenburg (1915). Foetuses were quickly removed from the anaesthetized female. Livers were excised, cut into small pieces and fixed by immersion in cold fixative. Samples of the liver taken from the newborn and adult animals were processed in the same way as the foetal ones. Catalase cytochemistry Experiments were carried out according to the technique introduced by Graham & Karnovsky (1966), with some modifications (LeHir, Herzog & Fahimi, 1979). Specimens werefixedin 2 % glutaraldehyde in 0-1 M-cacodylate buffer pH7-4, containing 5 % sucrose and 0-05 % CaCl2 for 30 min at 4 C and rinsed in the same buffer for approximately 1 h. Following a preincubation period of 30 min at 37 C in 0-1 M-Tris-HCl buffer pH 8-5, these were incubated at the same temperature and for different time intervals (30min-3h) in the same buffer containing 0-2% 3,3'-diaminobenzidine tetra-HCl (DAB) (Serva, FeinbiochemicaHeidelberg) and 0-2 % H2O2 (freshly added). In control experiments, preincubation and incubation media were supplemented with 0-1-0-2M-3-amino-l,2,4-triazole as an inhibitor of the peroxidatic activity of catalase. At the end of the incubation period, specimens were rinsed twice in distilled water for 10 min, three times in 0-1 M-cacodylate buffer pH 7-4 with 5 % sucrose for about 1 h andfinallypostfixed in 1 % OsO4 in 0-1 M-cacodylate buffer pH7-4 for 1 h at 4C. D-aminoacid oxidase cytochemistry Experiments were carried out according to the cerium technique introduced by Briggs et al. (1975) and adapted to the detection of peroxisomal oxidases in animal tissues by Veenhuis & Wendelaar Bonga (1977). Specimens, cut in blocks thinner than 1 mm to allow for the slow penetration rate of the cerous ions, were fixed by immersion in 1 % glutaraldehyde in 0-1 M-cacodylate buffer pH7-4 containing 5 % sucrose, lmM-CaCl2 and lmM-MgCl2 for 30 min at 4C. They were then rinsed three times in the same buffer for about 1 h. The blocks were preincubated for 30min at 37C in 0-lM-Tris-maleate buffer pH7-5 containing 5mM-cerium chloride and 0-lM-3-amino-l,2,4-triazole and then incubated at 37C for different time intervals (2-6 h) in the same medium containing 50 mM-D-alanine or Dproline (Fluka, Buchs, Switzerland). Controls were performed by incubating tissue in medium lacking substrate or by adding lOmM-kojic acid (D-AAO competitive inhibitor) both to the preincubation and incubation media. At the end of incubation, specimens were washed for 10 min in 0-1 M-cacodylate buffer pH6 to remove any cerium hydroxyde precipitate formed during incubation, then in 0-1 M-cacodylate buffer pH7-4 for about 1 h and postfixed in 1 % OsO4 in 0-1 M-cacodylate buffer pH7-4 for 1 h at 4C. After postfixation, all the specimens were dehydrated, embedded in Epon 812 and sectioned with a LKB Ultrotome III. These sections, unstained or stained with lead citrate and uranyl acetate, were observed under a Philips 400 T electron microscope. To obtain a rough idea of the developmenta (...truncated)