Cell Tissue Res (1985) 242:661-667

a n d rL. sue R e s e a f r . h �9 Springer-Verlag 1985

Morphometric studies on lipoprotein particles in developing rat liver and their corticosteroid-induced changes during the late gestational period H. Franke and R. Dargel Institute of Pathological Biochemistry, Friedrich Schiller University, Jena, German Democratic Republic

Summary. Changes in lipoprotein particles in hepatocytes of the fetal rat liver have been studied morphometrically from days 15-21 of gestation. On all these days, distinct lipoprotein particles are found within the cisternae of the RER, Golgi complexes and Golgi-derived secretory vesi- cles. Their mean diameter is 30-31 nm. The number of Golgi complexes per hepatocyte, the lipoprotein particle number per Golgi complex and the volume density of the latter remain unchanged within the developmental period examined. The volume density of lipid droplets, however, shows a significant decrease during this time.

Following corticosteroid treatment, the mean diameter of lipoprotein particles, the number of lipoprotein particles per Golgi complex, the volume density of the Golgi com- plex, and that of the lipid droplets increase significantly within the examined period, whereas the number of Golgi complexes per hepatocyte is reduced. These data support the view that triglyceride production in the fetal liver is directly or indirectly stimulated by corticosteroids admin- istered to the pregnant rat, thus giving rise to larger amounts of hepatic lipoproteins and lipids.

Key words: Fetal liver, rat - Corticosteroids - Golgi com- plex - Lipoproteins - Lipids

One of the principle functions of the liver is the synthesis of LP particles primarily those of the VLDL type, which appear as distinct spherical osmiophilic particles in the he- patocytes. The intracellular site of formation of VLDL par- ticles, their migration from ER cisternae to the Golgi com- plex, the compositional alterations during the transport to the cell border and the subsequent release into the circula- tion have been well documented in a series of integrated microscopic, biochemical and immunocytochemical investi- gations of the adult liver of the rat and other species (Alex- ander et al. 1976; Bouma et al. 1979; Claude 1970; Glau- mann et al. 1975; Hamilton et al. 1967; Hess et al. 1979; Jeejeebhoy and Breckenridge 1975; Jones et al. t967; Mah- ley et al. t969; Matsuura and Tashiro 1979; Ruderman

Send offprint requests to: Dr. H. Franke, Institute of Pathological Biochemistry, Friedrich Schiller University, DDR 6900 Jena, Ger- man Democratic Republic

Abbreviations: LP lipoprotein; VLDL very low density lipoproteins (d < 1.006 g/ml); RER rough endoplasmic reticulum ; ER endoplas- mic reticulum

et al. 1968). The structural differentiation of rat hepatocytes at different gestational periods has also been examined in detail (Chedid and Nair 1974; Daimon et al. 1982; De Wolf-Peeters et al. 1972; Dvorak 1971 ; Franke and Goetze 1963; Greengard et al. 1972; Herzfeld et al. 1973; Luzatto 1981 ; Sturgess and De la Iglesia 1972). To our knowledge, no morphological studies, however, have been reported concerning LP particles in the developing rat liver. In earlier studies, we have observed lipoprotein-like particles within Golgi cisternae, Golgi-associated vesicles and cisternae of the RER in fetal rat hepatocytes (Franke et al. 1982). Ap- plying the protein A-gold technique for immunocytochemi- cal labeling, these electron-dense particles have been identi- fied as containing apo B (Dfirer and Franke 1984).

The aim of the present study was to analyse, using mor- phometric methods, changes in hepatic LP in the developing rat liver during the gestational period from day 15 to day 21. As in the adult rat liver, the Golgi complex of fetal hepatocytes proved to be the most conspicuous site of the appearance of LP particles as a result of temporary storage. Therefore, we tried to quantify the LP by evaluating the number of Golgi LP particles, their size, and the volume density of the Golgi complexes. Furthermore, we treated pregnant rats with prednisolone in order to determine whether this compound has any effect on the LP particles in the fetal liver; glucocorticoids administered to rabbits and rats have been shown to cause hyperlipoproteinemia, a fatty liver and an increase in number and size of the intrahepatic LP particles (Brindley 1981; Hill and Droke 1963; Klausner and Heimberg 1967; Mahley et al. 1968; Reaven et al. 1974; Wiener et al. 1968).

Materials and methods

Wistar rats (Uje: Wist), approximately 3 months old, weighing 180-220 g, were colony-bred and housed under standardized conditions in our department. Males and es- trous females were mated overnight and the date of preg- nancy was determined by vaginal smears. The first day that sperm was present was counted as day one of gestation.

Exper imental conditions. For the morphometric analysis of the LP particles in fetal liver we chose days 15, 18 and 21 of gestation. 12 pregnant control rats and 12 corticoste- roid-treated animals were divided into 3 equal subgroups for liver tissue sampling on the aforementioned gestational days. Prednisolone administration was performed under

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similar conditions as described by Reaven et al. (1974). Each rat of the 3 subgroups received 5 intramuscular injec- tions of prednisolone acetate (0.6 mg/100 g body weight, VER Jenapharm, GDR). The first injection was given 8 days before day 15, 18, and 21 of gestation, respectively, the others being administered 3, 5, 6, and 7 days after the first injection. Rats of the control group received an equal volume (0.1 ml) of isotonic saline under the same conditions that prednisolone was administered. 24 h after the last injec- tion the rats were anesthetized for tissue sampling.

The liver tissue blocks (smaller than 1 mm 3) were fixed in phosphate-buffeted (0.12 M) 3% glutaraldehyde and subsequently postfixed in imidazol-osmium tetroxide ac- cording to Angermfiller and Fahimi (1982). This fixation procedure allowed good preservation of the LP particles in the fetal hepatocytes, thus facilitating their quantitative evaluation. Other cell structures, however, were less well fixed. The liver blocks were embedded in Micropal (Ferak, West Berlin), thin sections of approximately 60 nm (inter- ference colour: silver grey) were made and doubly stained with uranyl acetate and lead citrate (Reynolds 1963). For further technical details, see Franke et al. (1983).

Morphometry. From each pregnant rat, 3 fetuses were ran- domly dissected and, from each of those, 4 liver tissue sam- ples were prepared for morphometric analysis. The number of Golgi complexes per nucleated hepatocyte, the volume density of lipid droplets (Vvtip) and the Golgi complexes (Vvgot) and the number and size of Golgi LP particles were measured from 12 eIectron micrographs taken at random from each liver sample at 2 magnification levels: x 3000 and x 10000. All electron micrographs were photographi- cally enlarged 3 times, Morphometry was carried out using a coherent double lattice test system according to the rec- ommendations of Weibet (1979). The volume density of the Golgi complex was estimated according to the proce- dure of Sturgess and De la Iglesia (1972) and is expressed as cm3/cm a hepatocytic cytoplasm. The number of the Golgi LP particles -was quantified by counting all those particles located within Golgi cisternae and Golgi-asso- ciated secretory vesicles. The sizing of the LP particles was performed with a measuring magnifier device (VEB Zeiss, GDR). In each case~ at least 200 free-standing LP particles were measured, randomly chosen from liver samples of fe- tuses from the 3 subgroups of controls and prednisolone- treated rats.

All morphometric values were averaged per experimen- tal group, the group mean (-+ SEM) was calculated, and all data were subjected to statistical analysis (Student's t- test). In order to determine whether prednisolone affects the development of the rat fetuses we checked the weight of the liver and fetus of a total of 201 fetoplacental units on day 15 and 21 of gestation (104 fetuses from control rats and 97 from rats treated with prednisolone).

Results

Controls. On all days examined here, fetal hepatocytes con- tain a Golgi complex, which is very well-developed and frequently multiple In cross-section, the Golgi complexes are composed of an assembly of parallel, flattened cisternae with moderately dilated ends. At the trans-face of the Golgi stacks varying amounts of secretory vesicles can be seen.

Some of them and some of the Golgi cisternae contain LP particles (Figs. 1 a, 2). 11 is ofinlerest that fixation media known t o preserve LP particles in lhe adult rat liver ade- quately (Franke et at. t983, t985) do not stabiIize those of the fetal liver successfully; this may be a result of some difference in the biochemical composition of the fetal LP. RER is another prominent cellular compartment involved in LP metabolism. Within its cisternae single or multiple LP particles occur regularly during the gestational period from day 15 to day 21 (Fig. lb), On survey views of the fetal liver, a varying number of lipid droplets in the cyto- plasm is generally noticed; these are most numerous on day 15. On day 18 of gestation the first glycogen islets are visible.

Quantitative data. On day 15 of gestation a prominent peak of the size distribution pattern of LP particles is formed in the 31-34 nm range (Fig. 3). On day 18, the highest peak represents particles of the same size, whereas on day 21 there exist two, almost equally large, size classes comprising 23 27 nm and 27-31 nm particles, respectively. The mean diameter of LP particles on day 15 is 30 nm and that on days 18 and 21, 31 nm (Fig. 3). No significant changes are found in the developing hepatocytes regarding the volume density of the Golgi complex, the number of LP particles per Golgi complex, and the number of Golgi complexes per nucleated hepatocyte (Figs. 4, 5). The volume density of lipid droplets, however, is significantly reduced by 48% on day 18 compared with day ]5. A slight, but insignificant rise of ~7% folIows between days I8 and 2t of gestation (Fig. 4).

Prednisolone-treated group. Prednisotone treatment of preg- nant rats does not lead to a significant weight reduction of the fetuses. The fetal weight is 162+0.4 mg on day 15 and 3.51-1-0.06 g on day 21 in the control groups, and 156_+ 0.9 mg and 3~ 19 • 0.08 g, respectively, in the predniso- lone-treated groups. On the other hand, the fetal liver weight is reduced from 11 +0.2 mg to 6.0___0.3 mg (p <0.01) on day 15, and from 320 • 7.0 mg to 247 -I- 11 mg (p < 0.005) on day 21.

Prednisolone treatment of the pregnant rats is followed by a series of substructural alterations in the fetal liver in all 3 gestational periods examined. The Golgi complexes appear to be larger and their cisternae and vesicles are better filled with LP particles than in the controls (Figs. 6, 7). Moreover, assemblies of LP-containing vesicles occur in the hepatocytic cytoplasm; these are never detected in such amounts in the controls (Fig. 8). Similarly, LP particles are more numerous in the RER cisternae. In survey views of the fetal liver, the high incidence of lipid droplets (some even located in the cell nucleus) and frequent profiles of unusually large mitochondria are noteworthy findings. The bile capillaries are considerably dislended and contain fine- granular or larger lipidqike deposits (Fig. 8); these features are most marked on day 2~.

Quantitative data. Prednisolone treatment is followed by an increase in number and size of the fetal hepatic LP parti- cles at all gestational stages (Figs. 3 5). In comparison with the controls, the LP-particle diameter enlarges from 30 to 38 nm on day 15, from 31 to 37 nm on day 18, and from 31 to 36 nm on day 21 (Fig. 3). Thus, within the develop- mental period studied, prednisolone induces an increase in

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Fig. 2. Area of a hepatocyte, control group, demonstrating a Golgi complex (GA) on gestational day 21. The dilated ends of the Golgi cisternae and some vesicles (SV) are filled with LP particles (/~ ). rn Mitochondria, gl glycogen deposits. • 36000

Fig. 1. a Fetal rat liver, gestational day 15, control group, showing a Golgi complex (GA) whose cisternae and associated secretory vesicles (SI0 contain discrete electron-dense LP particles, m Mitochondria, N cell nucleus • 36000. b A single secretory vesicle (SI0 near RER cisternae with some LP particles ( s ) inside, x 46000

the part icle diameter by about 20%. Fur thermore , there is a prednisolone-mediated significant increases in the vol- ume density of the Golgi complex by about 79% on day 15, and by 44% and 57% on days 18 and 21, respectively (Fig. 4). Similarly, an increase in the volume density of the

int racytoplasmic lipid droplets has been recorded; this is highest on day 18 with an elevation of about 150%, whereas on days 15 and 21 the increase in the volume density of the lipid droplets is approximate ly equal at about 50%.

As is seen in Fig. 5, the LP particle content per Golgi

664

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Fig. 3. Frequency distribution of the diameter of Golgi LP particles in the developing rat liver on days 15, 18, and 21 of gestation in controls ( ) and prednisolone-treated animals ( . . . . . ). The arrows denote the mean particle diameter averaged from the differ- ent size classes

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Fig. 5. Number of LP particles per Golgi complex and number of Golgi complexes/100 nucleated hepatocytes in the developing rat liver of controls (C) and prednisolone-treated animals (PR) on days 15, 18, and 21 of gestation, x C versus PR is significant at p < 0.05 or less

Fig. 6. Fetal rat liver, gestational day 15, after prednisolone treatment of the mother. Hepatocytic area showing a Golgi complex (GA) and a few secretory vesicles (SV), partly densely filled with LP particles, m Mitochondria. x 35000

Fig. 7. Fetal rat liver, gestational day 21, after prednisolone treatment of the mother. Area of a Golgi complex (GA) with several secretory vesicles (SV) containing LP particles, (,~) RER cisternae with two LP particles inside, x 37000

Fig. 8. Juxtabiliary region of two hepatocytes, gestational day 21, after prednisolone treatment of the mother. The lumen of the bile capillary (BC) contains osmiophilic material. Both hepatocytes show tracks of transport vesicles (SV) containing LP particles. N cell nucleus, m mitochondria, GA Golgi complex, x 28 000

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complex is significantly augmented after prednisolone treat- ment of pregnant rats. On day 15, the LP particle number per Golgi complex increases from 35.8+_3.5 to 78.4+8.5, on day ~8 from 34.2+2.0 to 58.6+_t.5, and day 2I from 32.9_-t-3.5 to 56.2_+8.5. On the other hand, the number of Golgi complexes per nucleated hepatocyte is significantly reduced by 24% on day 15, whereas on days 18 and 21 we find only a reduction of 13%. Consequently, the number of LP particles per nucleated hepatocyte is increased to 167% on day 15, and to 148% on days 18 and 21.

Discussion

The results of the present microscopic studies provide evi- dence that, in fetal rats, a few days after the appearance of the liver primordmm on day 11 of gestation (Elias 1955; Monie 1965), LP particles appear within cell compartments shown to be closely involved in VLDL formation in adult rat liver (Franke et al, 1983, 1985; Hamilton et al. 1967; Hess et al. 1979; Jeejeebhoy and Breckenridge 1975; Jones etal. 1967; Mahley etal. 1969; Matsuura and Tashiro 1979). Our morphometric findings concerning the LP parti- cle number per Golgi complex indicate that, on day 15 of gestation, normal fetal hepatocytes have a Golgi LP- particle content that corresponds to the number determined in centrilobular hepatocytes of adult rat liver (Franke et al. 1983), Because the rates of synthesis and secretion, in com- bination with the turnover time, determine the hepatic LP pool, it is not possible to draw definite conclusions from the part~cIe number concerning the secretory activity of the liver.

Our morphometric data concerning the volume density of lipid droplets and the Golgi complex agree with those obtained by other authors in the fetal rat liver on days 15 and 18 of gestation (Daimon et al. 1982). The volume density of the intracytoplasmic lipid deposits shows a signif- icant decrease from days 15 to 18, at a time when the first glycogen islets begin to appear in the hepatocytes. During the following gestational days, the volume density of lipid droplets, however, remains nearly constant.

Although the estimation of the size of particle structures in tissue sections is difficult, electron micrographs enable LP particle profiles to be compared at different develop- mental stages. The LP particles in fetal rat liver have a mean diameter of only 30-31 nm. Consequently, they differ considerably in thear size from the Golgi LP particles in adult rat liver, where we have measured diameters of 48 nm in periportal and 45 nm in centrilobular hepatocytes (Franke et al, 1983) Our results here strongly suggest that fetal liver produces LP particles that can be considered from their size as VLDL and low density lipoproteins (LDL).

Prednisolone has been used in our experiments, since this hormone is known to modify hepatic glycerolipid me- tabolism (Brindley 1981 ; Cole el al, 1982; Heimberg et al. t9641 Klausner and Heimberg e1 al. 1964; Klausner and Heimberg ~9671 O~egovi6 et al. I975). On the other hand, no information is available concerning its action on hepatic LP of fetus when administered to pregnant rats. Interest- ingly, fetal rat liver responds to corticosteroid administra- tion in the same way as adult rat liver, a significantly ele- vated hepatic lipid and a rise in number and size of Golgi VLDL particles being observed (Reaven et al. 1974). The increase in the diameter of LP particles in the fetal hepato- cytes is, however, only about 20% in contrast to the adult

rat liver where a 32% enlargement of the particle size is obtaine& On the other hand, the prednisolone-induced ele- vation of the LP particle number per Golgi complex is high- er in feint liver than adult. Beside alterations of th~se struc- tures closely involved in LP metabolism, prednisolone treat- ment of pregnant rats ~s followed by a significant loss in fetal liver weight, a widening of the bile capillaries com- bined with the abnormal appearance of intrabiliary lipid- like deposits, Since in the fetal hepatocytes no structural lesions are detectable after prednisolone administration, we assume that the decrease in liver weight is mainly a result of an inhibition of the proliferation of hemopoietic cells; this is most intense on days 15 and 18 of gestation (Finck and Theil 1964).

The question remains unanswered whether the increased number of Golgi LP particles in the fetal liver is the result of a higher LP production or of a reduced particle release. The former provides the more suitable explanation. Corti- costeroids are known to mobilize lipids in white adipose tissue, thus leading to hyperlipacidemia, and to an increased hepatic uptake of fatty acids that stimulates hepatic trigly- ceride synthesis and hepatic VLDL output (for a review, see Brindley 1981), Since fetal rats have no appreciable lipid stores, the mobilization of maternal lipids may be of greater importance than any lipolylic processes in the fetus. The diaplacentar transport of free fatty acids guarantees a high- er input into the fetal liver, lhus lriggering triglyceride pro- duction, However, a direct glucocorlicoid effect on hepatic triglyceride syntheses in the fetus must also be taken into consideration. According te Glenny and Brindtey (1978), Jennings eta[. (t981) arid Lehtonen eta[. (1979) corticoids increase the activity of the phosphatidate phosphohydro- lase, thereby stimulating triglyceride production. In conclu- sion, we suggest that the prednisolone-induced increase in the total number of Golgi LP particles and in the volume density of lipid droplets is due to an elevated synthesis of triglycerides caused directly and indirectly by the glucocor- ticoid hormone.

Acknowledgements. The authors wish to express their thanks to Mrs. H. Guder, Mrs. K. Martin, Center for Electron Microscopy of the Medical Faculty, and Mr. F. Reim for valuable technical assistance.

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Accepted June 12, 1985