Histochemistry (1991) 96:7 12 0301556491000593 Histochemistry

© Springer-Verlag 1991

Immunocytochemical localization of albumin, transferrin, angiotensinogen and kininogens during the initial stages of the rat liver differentiation J.L. Gelly 1, J.P. Richoux 1, G. Grignon 1, J. Bouhnik 2, T. Baussant 2, F. Alhene-Gelas 2, and P. Corvol 2

1 Laboratoire d'Histologie-Embryologie, Facult6 de M6decine, B.P. 184, F-54505 Vandoeuvreqes-Nancy Cedex, France 2 INSERM U36, 17 rue du Fer-~-Moulin, F-75005 Paris, France

Received September 25, 1990 / Accepted February 25, 1991

Summary. Rat albumin, transferrin, angiotensinogen, T kininogen (TKg) and high molecular weight kininogen (HKg) gene expression was examined immunocytochem- ically in embryonic and fetal livers. All these plasmatic proteins, angiotensinogen excepted, are detected as early as day 11 of gestation in intestine epithelial cells and embryonic hepatocytes. Angiotensinogen becomes ex- pressible only at day 13 of gestation. During the early fetal period, the protein immunostaining increases strik- ingly in parallel with the hepatocyte differentiation. Al- bumin and transferrin are highly expressed comparative- ly to kininogens and angiotensinogen. For the first time, specific HKg is demonstrated in the rat liver.

Introduction

In adult mammals, the liver performs numerous physio- logical functions, the major one is the synthesis of plas- matic proteins. Although numerous immunocytochemi- cal data have reported their distribution in the adult rat liver (Feldmann et al. 1972, •975; Feldmann 1979; Foucrier et al. 1979; Morris et al. 1979; Yokota and Fa- himi 1981 ; Pignal et al. 1982; Richoux et al. 1983), only few developmental studies are available such as the im- munodetection of albumin (Nayak and Mital 1977; Dempo et al. •983), transferrin (Adamson •982) and some hepatic enzymes (Gaasbeek Janzen et al. 1988) at different stages of the rat or mouse development. Bio- chemical studies (Yeoh and Morgan 1974; Nayak and Mital •977; Yeoh et al. 1979; Adamson •982; Meek and Adamson •985; Williams et al. •986) have described the synthesis and the secretion of some plasmatic proteins with partial discrepancies probably due to the fetus dis- section and the lack of sensitive assays. In addition, albu- min and c~-fetoprotein m R N A levels have been reported in the rat liver from the 15th day of gestation (Liao et al. •980; Sellem et al. •984; Sell et al. 1985).

Offprint requests to: J.L. Gelly

Angiotensinogen and kininogens are the vasoactive peptide precursors of angiotensin II and bradykinin, re- spectively. In the rat, three kinds of kininogens (review in Nakanishi 1987) have been identified: LKg (low mo- lecular weight kininogen), HKg and TKg. LKg and HKg are encoded by the same gene and release brady- kinin under kallikrein hydrolysis. TKg, encoded by two highly homologous genes, is kallikrein resistant and re- leases Ileu-Ser-bradykinin (T kinin) only by trypsin ac- tion. These kininogens have a homologous heavy chain and differ only by their specific light chain. By immuno- cytochemistry, Utsunomiya et al. (•988) have reported TKg localization in the adult rat liver, but HKg has never been demonstrated.

In this report, we present the best immunocytochemi- cal conditions of detection of some strategic plasmatic proteins and the precise chronology of their gene expres- sion during the fetal liver development.

Materials and methods

Rat fetuses (Wistar strain) were dated from the discovery of sper- matozoons in the vaginal smears of the mated female (day 0 of pregnancy). The fetuses were collected from the 11th to the 14th day of gestation. Only M-day-old fetuses were decapitated before fixation,

Tissue preparation for irnmunocytochemistry

The fetal tissues were fixed for 15 min by immersion in different fixatives at 4 ° C to determine those which preserved the best immu- noreactivity. Bouin-Hollande fluid and freshly prepared paraben- zoquinone-paraformaldehyde (PBQ-PAF) solutions (0.4% para- benzoquinone, 1% or 4% paraformaldehyde supplemented with 5% sucrose in 0.1 M phosphate buffer, pH 7.4) were used.

The fetuses, fixed with Bouin-Hollande fluid, were dehydrated, embedded in paraffin and sectioned at 4 gm. Those fixed with PBQ-PAF solutions were rinsed in phosphate buffer, soaked at 4 ° C successively in 5%, 10% and 20% sucrose (W/V) in 0.1 M phosphate buffer, embedded in OCT compound (Lab. Tek. prod- ucts, Elkhart, USA) and freezed in liquid nitrogen cooled isopen- tane. Frozen sections, 5 I~m thick, were obtained with a cryostat and thawed into polylysine-coated slides.

Antisera

Rabbit anti-rat albumin and transferrin were purchased from Nordic Immunological Laboratories (Tilburg, The Netherlands).

Rabbit anti-rat angiotensinogen was characterized by Bouhnik et al. (1987). Rabbit anti-rat YKg and HKg were similar to those used by Baussant et al. (1988) and Suzuki et al, (1988). HKg LC specific (specific light chain of HKg) was obtained as described previously (Kerbiriou and Griffin 1979) and the specific antiserum was raised in the rabbit by the method of Vaitukatis et al. (1971).

Immunocytochemistry

The deparaffinized and frozen sections were rehydrated, equilibrat- ed with 0.1 M phosphate buffer (pH 7.4) containing Triton Jr-100 (0.5%), EDTA (1 mM) and lamb serum (1%).

The proteins were detected on serial sections for comparative studies. Briefly, the detection was carried out by successive incuba- tions in :

1) Specific antiserum for 2 h at 20 ° C (anti-albumin and -trans- ferrin: 1/20000th; anti-TKg, -HKg and -HKg LC specific: 1/ 1000th; anti-angiotensinogen: 1/200th) with cryostat sections. With paraffin sections, only TKg and HKg LC specific antisera dilutions are lower: 1/200th.

2) Biotinylated goat anti-rabbit IgG (Vector, Burlingame, USA): 1/250th for 45 min.

3) Preformed avidin biotinylated horseradish peroxidase com- plex (Vector) for 30 rain.

The peroxidase activity was selectively detected and intensified using diaminobenzidine 0.01%; Ni (SO,~)2 (NH,,)2, 0.02% and HzO2, 0.003% in phosphate buffer 0.1 M, pH 7.4. Kernechtrot was used for nuclear counterstaining.

Specificity of the immunostaining

The specificity of the immunostaining was established by inhibition tests using homologous antigens covalently linked to acrylamide- agarose beads (Ultrogel Ac A22 IBF, Paris, France).

Rat serum albumin-gel (0.8xl0-31aM/ml), transferrin-gel (0.03 p,M/ml), TKg-gel (1.4x 10 -3 gM/ml), HKg-gel (0.7x 10 -3 gM/ml) and angiotensinogen-gel (0.3 x 10 -3 gM/ml) were prepared.

Since TKg and HKg share a large homologous sequence in their heavy chain, a possible cross-reactivity, due to antibodies directed against common epitopes, was tested : TKg and HKg anti- sera were differentially adsorbed on HKg and TKg-gel respectively.

Figs. 1-6. Demonstration of the specificity of albumin (Figs. 1 and 2), transferrin (Figs. 3 and 4) and TKg immunostaining (Figs. 5 and 6) in 14-day-old rat fetal liver. Hepatocytes contain albumin (Fig. 1), transferrin (Fig. 3), and TKg (Fig. 5). Preabsorption of diluted specific antiserum with homologous protein-gel specifically abolishes the immunostaining (Figs. 2, 4 and 6). x 300

Results

The embryonic and fetal tissues cannot be fixed by per- fusion as per formed in the adult rats, consequent ly im- mersion-f ixat ion must be used. In the differentiating rat hepatocytes, the protein immunos ta in ing was condi- t ioned by the intracellular protein concent ra t ion and the fixative used. The best immunoreact iv i ty required a short tissue fixation in a 1% parabenzoqu inone-para fo r - maldehyde solution and cryosta t sectioning. Immers ion- fixation in more concent ra ted pa ra fo rmaldehyde solu- tions or in Bouin-Hol lande fluid induced a decrease or the complete disappearance o f immunoreact ivi ty . Never- theless, on account o f the small size o f l l - d a y - o l d rat

Figs. 7-10. Demonstration of TKg and HKg cross-reactivity in 14-day-old rat fetal liver. Numerous hepatocytes biosynthesize TKg (Fig. 7) and HKg (Fig. 9). Differential adsorption of TKg and HKg antisera with HKg- and TKg-gel respectively induces a significant decrease ofimmunostaining (Figs. 8 and 10). x 300

Figs. 11-14. Distribution of albumin (Fig. 11), transferrin (Fig. 12), TKg (Fig. 13) and HKg LC specific (Fig. 14) in 11-day-old fetal liver and foregut (Bouin-Hollande fixation and paraffin embed- ding). Immunoreactivity appears in the epithelial cells of the fore- gut (G) and the liver diverticulum (LD). High albumin staining (Fig. 11) is shown in all hepatocytes and numerous intestine cells whereas transferrin (Fig. 12) and kininogens (Figs. 13 and 14) stain- ing is restricted to a few cells, x 300

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Figs. 15-18. Differential expression of some plasmatic proteins in the fetal liver (age 14 days). All hepatocytes exhibit a strong albu- min and transferrin staining (Figs. 15 and 16, respectively) whereas

HKg LC specific (Fig. 17) and angiotensinogen (Fig. 18) are less expressed, x 300

embryos, a short Bouin-Hollande fixation and paraffin embedding allowed the preservation of protein immuno- reactivity and avoided the frequent liver primordium damage caused by cryostat sectioning.

When albumin (Figs. 1 and 2), transferrin (Figs. 3 and 4), TKg (Figs. 5 and 6), HKg and angiotensinogen antisera exhausted with homologous antigen-gel were used, the immunostaining was specifically abolished. In addition, TKg and HKg antisera cross-reacted signifi- cantly with the heavy chain of HKg and TKg respective- ly (Figs. 7-10), demonstrating the presence of antibodies directed against common epitopes of the heavy chain of these two molecules.

In 11-day-old embryos, the liver primordium is a ven- tral outgrowth from the foregut epithelium. Albumin, transferrin, TKg and HKg LC specific can be detected in the epithelial cells of the foregut and the liver diverti- culum (Figs. 11-14) but also in all entoblastic cells of the yolk salk. A strong albumin immunoreactivity can be shown in all hepatocytes and numerous intestine cells (Fig. 11) whereas transferrin (Fig. 12) and kininogens (Figs. 13 and 14) staining is restricted to a few cells. Angiotensinogen is exclusively detectable in the yolk salk epithelium.

At the 12th day of gestation, the hepatocytes are char- acterized by small size, thin processes and are organized into broad cords separated by large sinusoids. These cellular cords show an intense albumin and transferrin staining, a weak kininogen immunoreactivity and no an-

giotensinogen staining. One day later, angiotensinogen becomes detectable in a few hepatocytes and the staining intensity increases for all other proteins. At the 14th day of gestation, TKg (Fig. 7), HKg (Fig. 9) HKg LC specific (Fig. 17) and angiotensinogen (Fig. 18) are ex- pressed in an increasing number of hepatocytes whereas albumin (Fig. 15) and transferrin (Fig. 16) are present in all hepatocytes. These cells apparently contain less kininogens and angiotensinogen than albumin and transferrin.

Discuss ion

The present study is the first immunocytochemical dem- onstration of the precocious gene expression of some plasmatic liver proteins as albumin, transferrin, kinino- gens and angiotensinogen during the liver differentia- tion.

In the embryonic and fetal hepatocytes, neither col- chicine treatment, nor experimental biosynthesis en- hancement are required for protein accumulation as in the adult. In the adult rat liver, staining occurs as sparse granular formations in the Golgi apparatus and rough endoplasmic reticulum positions (Yokota and Fahimi 1981 ; Guillouzo et al. 1982; Pignal et al. 1982) whereas protein immunoreactivity is homogeneously distributed throughout the cytoplasm in the fetal hepatocytes.

11

In 11-day-old fetal liver, the plasmatic protein synthe- sis occurs as early as the hepatocytes differentiate f rom the foregut epithelium. The protein synthesis and storage take place in the epithelial cells of the foregut and the liver primordium. At this stage, all proteins studied are expressed, angiotensinogen excepted. In the liver primor- dium, albumin is the best marker of hepatocyte matura- tion since numerous embryonic hepatocytes are strongly stained whereas only a few express transferrin and kinin- ogens. Consequently, the biochemical gene expression of these proteins occurs before the morphological hepa- tocyte differentiation. Albumin and transferrin biosyn- thesis has been demonstrated in the rat and mouse yolk salk using radioimmunoassay (Yeoh and Morgan 1974), ELISA (Meek and Adamson 1985), [3H] leucine incor- pora t ion (Williams et al. 1986) and m R N A hybridiza- tion (Liao et al. 1980; Sellem et al. 1984). These data are supported by the observation that these two proteins are restricted to the entoblastic cells of the rat yolk salk as observed immunohistochemically at the 9th day of gestation (Shi et al. 1985; Gelly, unpublished results). Our results indicate that the hepatocytes accumulate al- bumin and transferrin as soon as these cells differentiate. This finding partially agrees with previous biochemical reports (Yeoh and Morgan 1974; Williams et al. 1986) who failed to detect transferrin and albumin before the 15th day and the 13th day of gestation respectively in the rat fetus. Between days 11 and 14 of gestation, the protein immunoreact ivi ty progressively increases in par- allel with the hepatocyte differentiation.

Our work provides the first immunocytochemical de- tection of H K g and T K g in the differentiating rat liver. The differential adsorption of H K g and T K g antibodies and the additional use of antibodies directed against the specific light chain of H K g allow to distinguish H K g from TKg. Apparently, the fetal hepatocytes contain more T K g than HKg. This finding is in a good agree- ment with the differential regulation of H K g and T K g gene expression in the adult rat (Nakanishi 1987). Angio- tensinogen is detected later than kininogens and its con- centration is the lowest of five proteins studied. This finding is consistent with the low level of angiotensino- gen m R N A in the fetal liver (Gomez et al. 1988).

Many functions have been ascribed to albumin: se- rum and interstitial fluid carrier for steroids, fatty acids, growth factors and other molecules. However, the con- cept of protein-regulated uptake of the various ligands has not been fully demonstrated (Siiteri e tal . 1982; Ockner et al. 1983). Transferrin binds iron and has mito- genic effets on the proliferating tissues (Meek and Adamson 1985). The strong albumin and transferrin synthesis in the embryonic and fetal hepatocytes suggests that these plasmatic proteins could be involved in the cell differentiation and growth. The precocious synthesis of kininogens and angiotensinogen supports the hypoth- esis that kallikrein-kinin and renin-angiotensin systems could play a role during the earliest phases of develop- ment.

Acknowledgements. This work was supported in part by a grant from the Bristol Myers Squibb Institut for medical research (Prin- cetown, New Jersey, USA) and the SDI CNRS n ° 1243.

References

Adamson ED (1982) The location and synthesis of transferrin in mouse embryos and teratocarcinoma cells. Dev Biol 91:227- 234

Baussant T, Michaud A, l~ouhnik J, Savoie F, Alhenc-Gelas F, Corvol P (1988) Effect of dexamethasone on kininogen produc- tion by a rat hepatoma cell line. Biochem Biophys Res Commun 154:1160-1165

Bouhnik J, Baussant T, Savoie F, Alhenc-Gelas F, Gauthier F, Esnard F (1987) Direct radioimmunoassay for rat T-kininogen. Biochem Biophys Res Commun 144:1090-1097

Dempo K, Sasaki M, Kaku T, Satoh M, Oyamada M, Mori M (1983) Immunohistochemical studies on e-fetoprotein and albu- m.in containing cells in the liver during ontogenesis and early stage of 3'-ME-DAB hepatocarcinogenesis. Ann NY Acad Sci 417:195-202

Feldmann G, Penaud-Laurencin J, Crassous J, Benhamou JP (1972) Albumin synthesis by human liver ceils: its morphologi- cal demonstration. Gastroenterology 63 : 1036-1048

Feldmann G, Maurice M, Sapin C, Benhamou JP (1975) Inhibition by colchicine of fibrinogen translocation in hepatocytes. J Cell Biol 67 : 237 243

Feldmann G (1979) Morphological aspects of hepatic synthesis and secretion of plasma proteins. In: Popper H, Schaffner F (eds) Progress in liver diseases, vol 6. Grune and Stratton, New York, pp 23-41

Foucrier J, Kraemer M, Vassy J (1979) D6monstration ultrastruc- turale de la synth~se de la transferrine dans le foie de rat adulte. CR Acad Sci Paris 288 : 1195 1198

Gaasbeek Janzen JW, Westenend P J, Charles R, Lamers WH, Moorman AFM (1988) Gene expression in derivatives of em- bryonic foregut during prenatal development of the rat. J Histo- chem Cytochem 36:1223-1230

Gomez RA, Cassis L, Lynch KR, Chevalier RL, Wilfong N, Carey RM, Peach MJ (1988) Fetal expression of the angiotensinogen gene. Endocrinology 123:2298-2302

Guillouzo A, Beaumont C, Le Rumeur E, Rissel M, Latinier MF, Guguen-Guillouzo C, Bourel M (1982) New findings on im- munolocalization of albumin in rat hepatocytes. Biol Cell 43:163-172

Kerbiriou DM, Griffin JH (1979) Human high molecular weight kininogen: studies of structure-function relationships and of proteolysis of the molecule occurring during contact activation of plasma. J Biol Chem 254:12020 12027

Liao WSL, Conn AR, Taylor JM (1980) Changes in rat el-fetopro- tein and albumin mRNA levels during fetal and neonatal devel- opment. J Biol Chem 255:10036-10039

Meek J, Adamson ED (1985) Transferrin in foetal and adult mouse tissues: synthesis, storage and secretion. J Embryol Exp Mor- phol 86:205-218

Morris BJ, Iwamoto HS, Reid IA (1979) Localization of angioten- sinogen in rat liver by immunocytochemistry, Endocrinology 105 : 796-800

Nakanishi S (1987) Substance P precursor and kininogen: their structures, gene organizations and regulation. Physiol Rev 67:1117-1142

Nayak NC, Mital I (1977) The dynamics of c~-fetoprotein and albu- min synthesis in human and rat liver during normal ontogeny. Am J Pathol 86:359-374

Ockner RK, Weisiger RA, Gollan JL (1983) Hepatic uptake of albumin-bound substances: albumin receptor concept. Am J Physiol 245G 13 : 6-18

Pignal F, Maurice M, Feldmann G (1982) Immunoperoxidase lo- calization of albumin and fibrinogen in rat liver fixed by perfu- sion or immersion : effect of saponin on the intracellular pene- tration of labeled antibodies. J Histochem Cytochem 30:1004- 1014

Richoux JP, Cordonnier JL, Bouhnik J, Clauser E, Corvol P, Men- ard J, Grignon G (1983) Immunocytochemical localization of angiotensinogen in rat liver and kidney. Cell Tissue Res 233:439-451

Sell S, Longley MA, Boulter J (1985) ~-fetoprotein and albumin

12

gene expression in brain and other tissues of fetal and adult rats. Dev Brain Res 22:49-53

Sellem CH, Frain M, Erdos T, Sala-Trepat JM (1984) Differential expression of albumin and e-fetoprotein genes in fetal tissues of mouse and rat. Dev Biol 102:51-60

Shi WK, Hopkins B, Thompson S, Heath JK, Luke BM, Graham CF (1985) Synthesis of apolipoproteins, alphafoetoprotein, al- bumin and transferrin by the human foetal yolk sack and other foetal organs. J Embryol Exp Morphol 85:191-206

Siiteri PK, Murai JT, Hammond JL, Nisker JA, Raymoure WJ, Kuhn RW (1982) The serum transport of steroid hormones. Recent Prog Horm Res 38:452510

Suzuki H, Alhenc-Gelas F, Bouhnik J, Corvol P, Menard J (1988) Differential effects of nephrectomy and surgery on plasma kin- inogens and angiotensinogen in the rat. Endocrinology 122: 2809-2815

Utsunomiya I, Oh-Ishi S, Hayashi I, Maruhashi J, Tsuji N, Yama-

moto N, Yamashina S (1988) Monoclonal antibodies against rat T-kininogen: application to radioimmunoassay and immu- nohistochernistry. J Biochem i03:225-230

Vaitukatis J, Robbins JB, Nieschalag E, Ross GT (1971) A method for producing specific antisera with small doses of immunogen. J Clin Endocrinol Metab 33:988-999

Williams CL, Priscott PK, Oliver IT, Yeoh GCT (1986) Albumin and transferrin synthesis in whole rat embryo cultures. J Em- bryol Exp Morphol 92:33-41

Yeoh GCT, Morgan EH (1974) Albumin and transferrin synthesis during development in the rat. Biochem J 144:215-224

Yeoh GCT, Wassenburg JA, Edkins E, Oliver IT (1979) Synthesis and secretion of albumin and transferrin by foetal rat hepato- cyte cultures. Biochim Biophys Acta 565:342355

Yokota S, Fahimi HD (1981) Immunocytochemical localization of albumin in the secretory apparatus of rat liver parenchymal cells. Proc Natl Acad Sci USA 78:4970-4974