Immunochemical investigations on the origin of serum albumin in … · Immunochemical investigations on the origin of serum albumin in the chick embryo ByD. ZACCHEO1 & C. E. GROSSP

/ . Embryol. exp. Morph. Vol. 18, 2, pp. 289-98, October 1967 2 8 9With 6 plates

Printed in Great Britain

Immunochemical investigations on the origin ofserum albumin in the chick embryo

ByD. ZACCHEO1 & C. E. GROSSPFrom the Institute of Anatomy, University of Genoa

Information concerning physical and chemical properties of serum albuminduring the development of the chick has been collected by different techniques,e.g. paper, gel and continuous flow electrophoresis, ultracentrifugation andimmunochemical methods. However, the results of the various authors arenot always in full agreement. The main questions deal with the precise stage ofdevelopment when serum albumin can first be detected in the circulatingblood, and with the heterogeneity of the molecular types in the group of serumalbumins. Other aspects of the problem concern the possibility of differentorigins of serum albumin at various stages and the metabolism of this proteinwhich probably has peculiar features at each intra- and extra-ovular develop-mental period (Ivanyi, Hraba & Cerny, 1964).

The stage at which serum albumin can first be detected in the circulatingblood or in embryonic extracts has been indicated as ranging between the 3rdand the 10th day of incubation (Nace, 1953; Weller & Schechtman, 1962).These differences are probably related both to the molecular types which havebeen investigated and to the techniques which have been employed.

Hepatic synthesis of serum albumin has now been demonstrated in mammals(Hamashima, Harter & Coons, 1964) and it is also commonly admitted forbirds; in the latter, however, a direct demonstration is still lacking and, inparticular, it is unclear when hepatocyte synthesis of serum albumin begins.

Our research has dealt with two of the above-mentioned problems, i.e.(1) when can serum albumin first be demonstrated in the serum and (2) whendoes the hepatic synthesis of this protein begin? We have used immunochemicaltechniques (Ouchterlony test and immunoelectrophoresis), taking as a referencethe serum albumin of the adult. For our embryonic stages we shall speak ofimmunochemically adult-like molecules, that is of molecules with, at least, thesame antigenic determinants.

As an additional criterion of the function of the hepatocytes at differentstages of development, the appearance of hepatic glycogen has been investigatedboth by chemical and by histochemical methods. Sections of liver of embryos

1 Author's address: Institute of Anatomy, Cagliari, Italy.2 Author's address: Institute of Anatomy, University of Genoa, Genoa, Italy.

290 D. ZACCHEO & C. E. GROSSI

and of various stages after hatching have also been examined by techniquesfor the histochemical demonstration of acid phosphatase and succinic oxidaseactivities.

MATERIAL AND METHODS

Preparation of antisera. We have used two types of antiserum. The first,prepared against total adult chicken serum (anti-TACS), has been suppliedby Behringwerke; the second, against adult chicken serum albumin (anti-CSA),was prepared in our laboratory from two groups of rabbits. The first group offive animals received 40 mg of albumin intramuscularly; 5 weeks later, afteradministration of an anti-hystaminic drug, 10 mg of the protein was injectedintravenously. A test bleeding was performed 1 week after the last injectionand in the event of a good antibody titre (a-precipitation test), total bleedingwas effected. When the antibody response was insufficient a recall dose wasinjected (10 mg of the protein intravenously once a month). A second group offive animals was immunized by interscapular injection of 70 mg of the proteinin 3 ml, together with an equal amount of complete Freund's adjuvant. Onemonth later a bleeding test was performed and, in the event of an insufficientantibody titre, a recall dose, identical to the first, was injected. An antibodytitre of 1/32,768 (a-precipitation test) was considered satisfactory. The seraof the two groups were separately pooled.

The antigen was prepared from adult chick serum. Globulins were firstprecipitated at 50 % saturation of ammonium sulphate; the supernatant wasdialysed for 7 days against repeated changes of phosphate-buffered saline andconcentrated in PVP (M.W. 25000, 25% solution). The protein was thenfiltered through Sephadex G-200 (Pharmacia) using phosphate-buffered salineas eluent and the filtrate collected in 5 ml fractions. The albumin peak waslocalized by spectrophotometric reading at 280 m/t. A second concentrationof the protein was thereafter performed by PVP up to 10 mg/ml (micro-Kjeldahltechnique). Anti-CSA and anti-TACS sera, when tested in Ouchterlony platesagainst this antigen, gave a single precipitation line.

Collection of chick sera. Blood was collected from vessels of the amniotic sacof the embryos, from the heart in young chickens and from the carotids in theadults. Sera were stored at -20 °C (without preservative) and thawed onlyonce before use.

Amniotic and allantoic fluids were collected from the sacs after isolation ofthe embryo with its whole membranes. Both fluids were tested with anti-CSAand anti-TACS sera.

Preparation of a protein fraction from yolk. For the preparation of proteinextracts we have applied the method of Shepard & Hottle (1949), using yolkobtained from fresh non-fertile eggs. Yolk was diluted 1:2 with saline, filteredthrough gauze and extracted with ether 6 times at 0 °C. The residual materialhas been centrifuged at 10000 g for 15 min. This procedure allows the separation

Origin of chick serum albumin 291

of a supernatant containing lipids, an intermediate fluffy layer and, at thebottom, a clear protein solution. This has been tested with anti-CSA and anti-TACS sera.

Fractionation and extraction of liver homogenates. Liver homogenates wereprepared in 0-25 M sucrose at 0 °C using a Potter-Elvehjem type homogenizerwith Teflon pestle. The livers were collected every other day from the 6th dayof incubation up to hatching; in addition, livers from chickens, non-laying hensand adults were used.

From the homogenate the following fractions have been prepared by centri-fugation (at 0 °C): nuclear fraction at 2000 g for 5 min; mitochondria at 3300gfor lOmin;lysosomes at 10 000 g for 25 min; microsomes at 25 000 gfor 120 min.Since the histochemical tests for acid phosphatase indicate the almost completeabsence of lysosomes in the hepatocytes (as shown in livers of embryos at the6th day of incubation (Plate 1)), centrifugation to recover the lysosomal fractionwas omitted in this case.

A morphological control of the fractions was not performed; however, thefractionation of the homogenate was checked through the assay of someenzymatic activities bound to cytoplasmic organelles. It has thus been shownthat acid phosphatase activity was detectable in lysosomal fractions while itwas lacking in the lighter fractions. On the other hand, only the microsomalfractions exhibited glucose-6-phosphatase activity, which was never present inthe heavier fractions.

The nuclear as well as the soluble fractions have not been considered in thisstudy. The mitochondrial, lysosomal and microsomal fractions were washedonce in 0-25M sucrose and then dissolved in £ of the original volume of thehomogenate of 1 % Triton X-100 (Rhom and Haas) in saline. In using TritonX100 instead of the more commonly used sodium deoxycholate we havefollowed the suggestion of Ugazio (1962a, b). In fact, unlike some ionicsubstances, a non-ionic detergent avoids the formation of non-specific precipi-tation lines in the agar gel (Tombs & Weston, 1961). Extraction (for at least30 min at 0 °C) was followed by centrifugation to sediment the undissolvedparticles. The nitrogen content of each solution has been standardized at around0-40 mg/ml (micro-Kjeldahl). The protein solutions thus prepared were usedfor the immunological tests.

In the case of adult chicken, mitochondrial, lysosomal and microsomalproteins have also been extracted from fractions of kidney homogenates.

Glycogen determination. The chemical determination of the glycogen contenthas been accomplished on 5 % homogenates in 0-15 M KC1, 0-1 M KF (Zaccheo,Orunesu & Grossi, 1963), following the method of Van der Kleji (1951). Forthe histochemical demonstration of the polysaccharide, tissue sections preparedfrom cold Gendre-fixed material were treated for the PAS-reaction with andwithout saliva digestion.

Enzyme-histochemical methods. Succinic oxidase activity (Plate 2) was


demonstrated by the Nitro-BT technique (Pearse, 1960). An excellent demon-stration of acid phosphatase activity was obtained with Gomori-type substrate,prepared as suggested by Holt & Hicks (1961).

RESULTS

The immunoelectrophoretic patterns obtained with chicken sera tested againstrabbit anti-CSA and anti-TACS sera have shown that serum albumin can bedemonstrated in embryos at the 4th day of incubation (Plate 3). A singleprecipitation line, in the albumin zone, characterized the immunopherograms.It must be noted that, in two specimens of serum from embryos at the 4th day,albumin showed a different pattern of migration (Plate 3). The identificationof the protein was accomplished by comparing the precipitation line obtainedin Ouchterlony plates by the use of anti-TACS serum with the line whichappears in the albumin-anti-albumin system. The continuity of the two lineswas considered as a reaction of identity. Only sera from 10-day embryos showeda second precipitation line in p-zone; this may be taken as an indication ofa protein which migrates faster than albumin and can be considered a pre-albumin (Plate 3). After the 14th day of incubation, the adult-type pattern ofthe precipitation band for albumin is established; the albumin-anti-albuminline appears in its characteristic boat-shaped form (Plate 4). An inspection ofthe series of immunopherograms of sera in different stages of developmentshows the gradual establishment of the protein pattern.

The amniotic fluid contains serum proteins from the 4th day of incubation.The combined use of anti-CSA and anti-TACS shows that, at least in stagesbefore the 14th day, only serum albumin is present (Plate 5, A). Quite similarresults have been given by the allantoic fluid in which serum albumin has alsobeen demonstrated (Plate 5, A). However, the intensity of the precipitationlines has always been very weak.

In protein extracts of yolk from non-fertile eggs we have detected at leastsix proteins which immunochemically correspond to those of the adult serum;among these, there is an albumin with serum-like characters (Plate 5, B).

Serum proteins and, in particular, albumin, were never demonstrated inextracts of liver mitochondria at any developmental stage. The lysosomalextract prepared from the 8th day of incubation always gave, in Ouchterlonyplates, a single precipitation line both with anti-CSA and anti-TACS serum(Plate 5, C). The reaction of identity for the two lines indicates that serumalbumin is present in the lysosomal fraction. The microsomal extract preparedfrom liver at the 6th day of incubation did not give any reaction with the immunesera. On the other hand, starting from the 8th day a precipitation line with themicrosomal extract has been demonstrated and identified as the result of analbumin-anti-albumin reaction. This line was always weaker than the oneobtained with the lysosomal extract. The immunochemical identity of lysosomal

J. Embryol. exp. Morph., Vol. 18, Part 2 PLATE

Acid phosphatase activity in liver sections of embryos at the 4th day (1), 6th day (2), 8thday (3), 14th day (4), hatching (5) and of adult chick.

D. ZACCHEO & C. E. GROSSI facing p. 292

J. Embryo!. exp. Morph., Vol. 18, Part 2 PLATE 2

51HSSSI

Succinic oxidase activity in liver sections of embryos at the 6th day (1), 8th day (2), 12thday (3), 20th day (4) and of chicken at 21 days (5) and 45 days (6).

D. ZACCHEO & C. E. GROSSI

J. Embryo/, exp. Morph., Vol. 18, Part 2 PLATE 3

4th

4th

8th

10th

12th

14th

Immunoelectrophoretic patterns obtained for chicken sera by the use of anti-TACS seraduring early embryonic stages.


/ . Embryol. exp. Morph., Vol. 18, Part 2 PLATE 4

16th

20th

Hatching

1st

5th

Adult

Immunoelectrophoretic patterns obtained for chicken sera by the use of anti-TACS seraduring late embryonic stages and after hatching.

D. ZACCHEO & C. E. GROSST

J. Embryol. exp. Morph., Vol. 18, Part 2 PLATE 5

Embryo 8th

Anti-CSA

•

Amn. • # All.

Anti#TACS

Amn. • • All.

•Anti-CSA

Adult

Anti-CSA a-TACS a-CSA a-TACS

Mt. Ms.

A. Demonstration of serum albumin in amniotic and allantoic fluids.B. Tmmunoelectrophoresis of a yolk-protein fraction against anti-TACS serum.C. Demonstration of serum albumin in lysosomal (Ly.) and microsomal (Ms.) extracts fromliver homogenates at different developmental stages. Mitochondrial extracts (Mt.) are negative.


/ . Embryol. exp. Morph., Vol. 18, Part 2 PLATE 6

Embryo 8th Hatching Adult

Anti-CSA

Ms.

Anti-TACS

Anti-CSA

• • Kidney Mt.

• Anti-CSA

• # Liver Mt.

% % Kidney Ly.

• Anti-CSA

• 9 Liver Ly.

• • Kidney Ms.

• Anti-CSA

• # Liver Ms.

A. Reaction of identity for lysosomal (Ly.) and microsomal (Ms.) albumin.B. A comparison of the results obtained in agar gel diffusion tests for kidney and livermitochondrial (Mt.), lysosomal (Ly.) and microsomal (Ms.) extracts.

D. ZACCHEO & c. E. GROSSI facing p. 293

Origin of chick serum albumin 293and microsomal albumin can also be demonstrated (Plate 6, A). Hence it canbe concluded that from the 8th day of incubation a molecule immunochemicallycorresponding to serum albumin can be demonstrated in liver extracts. Extractsprepared from kidney of adult chicken never gave any reaction with immunesera (Plate 6, B).

Glycogen determinations have shown a detectable amount of this poly-saccharide only from the 8th day of incubation. The values increase progressivelyup to hatching (Text-fig. 1). Histochemical tests confirm these results.

5 6 7 10 13 16 18Days of incubation

21 5

Text-fig. 1. Biochemical determination of hepatic glycogen in differentdevelopmental stages.

With the histochemical reactions for the succinic oxidase activity, manymitochondria in the hepatocytes are stained on the 4th day of incubation; thestained mitochondria increase in number during further development (Plate 2).

A positive reaction for acid phosphatase has been noted in Kupffer's cellsin embryos on the 4th day. A few lysosomes have been demonstrated withinthe hepatocytes on the 6th day, but their number undergoes a definite increaseonly from the 8th day (Plate 1).

DISCUSSION

Before discussing the results presented in this paper, it seems advisable toconsider the immunochemical techniques which have been employed, especiallyin relation to the occurrence of artefacts such as those occurring because ofcontamination of cell fractions by serum proteins (Perlmann, Hultin, D'Amelio


& Morgan, 1959; D'Amelio & Perlmann, 1960; D'Amelio, Mutolo & Piazza,1963).

The first question is the possibility that the albumin which has been demon-strated in lysosomes and microsomes might have been adsorbed during thepreparation of the homogenates. Further, in view of the fact that lysosomalproteins have, on the whole, a basic character (de Duve, 1963), we have con-sidered the possibility of an electrostatic interaction with albumin, whose iso-electric point is in the acid range. Perfusion of embryonic livers being practicallyimpossible, the cell fractions were washed in 0-25 M sucrose before extraction.

An immunological control like that of D'Amelio & Perlmann (1960) couldnot be performed because extracts of embryonic liver yield a concentration ofantigen too low for a proper preparation of immune sera. However, thepossibility of an interaction in vitro between lysosomes or microsomes and anacid serum protein (bovine serum albumin) could be ruled out by immunochemi-cal analysis. Other observations add further to the reliability of our findings.In the 6-day embryos serum albumin, which is already present in the circulatingblood, could not be detected in the extract of liver microsomes; furthermore,mitochondrial, lysosomal and microsomal extracts from adult chicken kidneyhave constantly been found to be devoid of albumin. Finally, the fact that ofthe various serum proteins only albumin has been detected speaks also againsta contamination.

Our results show that a molecule having the same antigenic determinantsas serum albumin is detectable in the circulating blood already on the 4th dayof incubation. As previously noted, the first appearance of albumin in serumhas been recorded within wide limits. Generally those authors who, like Nace(1953), have used immunochemical techniques (ring-test after Boyd), indicatean earlier appearance than those who, like Heim & Schechtman (1954) andWeller & Schechtman (1962), have applied physical methods (paper and con-tinuous flow electrophoresis).

These discrepancies can be attributed both to the different sensitivities of themethods and to possible modifications which the same type of molecules canundergo in some of its physical properties during development (Marshall &Deutsch, 1950; Schjeide & Deutsch, 1953).

As to the origin of serum proteins, Nace (1953) has proposed a theory whichmay apply also to albumin. The occurrence of cross-reactions between yolkand anti-TACS serum and the results of different adsorptions suggest, accordingto this author, that in the molecular population of albumin one can distinguisha vitelline from a non-vitelline fraction, the latter probably being synthesizedwithin the body of the embryo. The possibility of a transfer of protein moleculesfrom yolk to the circulating blood has also been accepted by Schechtman (1947).Circumstantial evidence for the utilization of yolk proteins has been given byMclndoe (1960) who has recorded, up to the 14th day of incubation, a gradualdecrease of the protein nitrogen of the yolk. Both Schechtman and Nace, adapt


the hypotheses of Weiss (1947) and Tyler (1947) to this problem and suggestthat a transfer of proteins from yolk to serum means something more thana simple supply to an organism not yet able to synthesize them. They thinkthat, through the yolk, a transfer of maternal molecules (accumulated duringoogenesis) to the embryo occurs; these molecules could act either as stimulantsfor an autonomous synthesis or as templates from which new molecules couldbe formed.

Our immunoelectrophoretic investigations on yolk have shown the presenceof at least six proteins, albumin among these, which have immunochemicalcharacters like the corresponding serum proteins of the adult.

A transfer of serum proteins to the yolk during oogenesis has also beendemonstrated in many animal species by immunohistological techniques(Knight & Schechtman, 1954; Kaster & Schechtman, 1957; Telfer, 1958,1961;Glass, 1959 a, b, 1961, 1962, 1963; Mancini et al. 1961, 1962, 1963; Telfer &Melina, 1963).

In view of the demonstration that yolk proteins can reach the embryonicserum unchanged (Brierly & Hemmings, 1956) one should accept the immuno-chemical identity of yolk molecules and serum molecules either before or afterthe period when embryonic synthesis begins.

The results lead to the conclusion that the embryonic liver begins to synthesizealbumin between the 6th and 8th days of incubation. This accounts for thepresence of albumin in microsomal extracts.

The presence of serum albumin in lysosomal extracts is more difficult tointerpret. It can be hypothesized that albumin in lysosomes represents ametabolic fraction which is taken by pinocytosis from serum in the liver cellsto be degraded by the proteolytic enzymes (cathepsins) which are a part of thelysosomal enzymic complement.

It should be mentioned that a labelled heterologous albumin has been detected,after parenteral administration, in the lysosomal fraction of mouse liver (Mego& McQueen, 1965) and that a highly active metabolic system is necessary fora protein that, like serum albumin, has a rather short half-life (from 1-75 to2-65 days, according to the determinations of Patterson et al. 1962). If lysosomesplay any role in the metabolism of serum albumin it must be assumed thatbefore the 8th day of incubation when a lysosomal system has not yet appeared,a different metabolic pathway is followed. The presence of albumin in theallantoic and amniotic fluids, the latter also before sero-amniotic junctionoccurs, may help in the interpretation of this question.

Experiments on surviving rat liver (Roberts & White, 1949) suggest thatglyconeogenesis from serum albumin may occur in vitro. Livers depleted ofglycogen can synthesize in vitro remarkable amounts of glucose while at the sametime albumin disappears from the medium.

In the liver of the chick embryo between the 6th and 8th days of incubation,we have shown that the hepatocyte acquires a number of important properties:


namely, the capacity for albumin synthesis, the differentiation of a lysosomalsystem and the beginning of glycogen storage. The latter facts might perhapsbe related to the onset of a new metabolic pathway for serum albumin andsuggest that, in previous ontogenetic periods, the protein derived from yolkmight be employed in a glyconeogenetic cycle.

SUMMARY

1. The origin of serum albumin has been investigated during the developmentof the chick.

2. A protein with the antigenic determinants of adult serum albumin canalready be detected in embryos at the 4th day of incubation.

3. Serum albumin can be demonstrated in liver microsomal extracts onlyfrom the 8th day of incubation. In the same period it appears also in liverlysosomal extracts.

4. Albumin is present in yolk, in the amniotic fluid even before the sero-amniotic junction is established, and in the allantoic fluid.

5. It is suggested that, up to the 6th day of incubation, serum albumin issupplied to the embryo from the yolk-reservoir and that, after the 6th day, itcan also be synthesized in the liver.

6. A correlation has been attempted for the beginning of albumin synthesiswithin the hepatocyte, the beginning of glycogen storage and the differentiationof a lysosomal apparatus. All these occur between the 6th and the 8th day ofincubation.

RESUME

Etudes immunochimiques sur Vorigine de la seralbuminechez Vembryon de poulet

1. L'origine de la seralbumine a ete essayee pendant le developpement del'embryon de poulet.

2. Une proteine avec les determinants antigeniques de la serumalbuminede l'Adulte peut etre demontree dans le sang a partir du 4eme jour d'incubation.

3. On a pu demontrer la presence de serumalbumine dans les extraits lyso-somaux du foie seulement apres 8 jours d'incubation.

4. L'albumine est presente dans le vitellus, dans le liquide amniotique, memeavant la communication serum-amniotique, et dans le liquide allantoiidien.

5. On a suggere que, jusqu'au 6eme jour d'incubation la serumalbumineparvient a l'embryon de vitellus et que, apres, cette proteine peut etre synthetiseedans le foie.

6. On a cherche de trouver une relation entre la synthese hepatique de l'albu-mine, Paccumulation du glcyogene et la differentiation d'un appareil lysosomal.Tous ces faits se verifient dans une periode comprise entre le 6eme et le 8emejour d'incubation.


This investigation has been supported by the Italian Consiglio Nazionale delle Ricerche.

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(Manuscript received 3 April 1967, revised 29 May 1967)

Documents

Immunochemical investigations on the origin of serum albumin in … · Immunochemical investigations on the origin of serum albumin in the chick embryo ByD. ZACCHEO1 & C. E. GROSSP