Albumen absorption during chick embryogenesis

Indeed the major part of albumen is very likely absorbed across the yolk and the amnion. In this connexion the function of the albumen sac is still not clearly known. This sac, formed starting from the 9th to 10th day of incubation by a folding of the chorioallantois, is considered by some authors to be responsible for albumen absorption (Patten, 1950).

During development, the protein composition of the residual albumen remains substantially unchanged and very similar to that of the non-incubated egg (as judged by paper and free-boundary electrophoresis) (Marshall & Deutsch, 1950; Rizzoli, 1956); besides, the immunological behaviour of the individual proteins is unmodified (Kaminski & Durieux, 1954). The electrophoretic data indicate also that the relative proportion of different albumen proteins is constant during incubation (Marshall & Deutsch, 1950), though some quantitative observations demonstrate a relative diminution in ovalbumin concentration (Hasegawa, Taguchi & Hasegawa, 1956).

Less is known about the protein composition of amniotic fluid (Kaminski & Durieux, 1954; Geelhoed & Conklin, 1966). With regard to the yolk it has been demonstrated that the albumen proteins can be recovered from the water soluble fraction (WSF) (Carinci & Manzoli-Guidotti, 1968).

Up to the present time,however,thereis no analysis performed simultaneously on embryonic fluids to furnish a complete picture of the qualitative and quanti-tative modes of albumen absorption. In this research we have followed the albumen absorption, determining at the same time the wet weight, dry weight and protein content of residual albumen and amniotic fluid, and also estimating relative proportion of yolk WSF proteins. In addition we have carried out electrophoretic and ultracentrifugal analysis of the proteins of these embryonic fluids.

We have used eggs (60 ± 0’5 g, average weight) laid by a small group of White Leghorn hens in June-October 1967, provided by the Corticella agricultural station (Bologna). The eggs were incubated at 38 ± 0·5 °C, at 60 % relative humidity and turned twice a day.

Fluids were obtained from fertile non-incubated eggs within a 24 h period of their being laid (albumen and yolk) and also after 10, 12, 13, 14, 15, 16, 17 and 18 days of incubation (albumen, yolk and amniotic fluid), utilizing at least 10 eggs per day, and placed according to the embryo morphogenetic age, evaluated by Hamburger-Hamilton development stages (Hamilton, 1952).

Albumen was removed in toto, including the chalazae, immediately weighed and diluted with 0·16 M NaCl (1:2, w/w) to obtain an albumen dilution without ovomucin precipitation (Forsythe & Foster, 1950); then the albumen was homo-genized with a Waring blender. From this material globulins were prepared by repeated precipitations with ammonium sulphate at 45–50 % saturation (Carinci & Manzoli-Guidotti, 1968); ovomucoid was prepared according to Warner (1954). At 10 and 12 days of incubation, owing to the difficulty of completely isolating albumen from yolk sac, quantitative determinations were also carried out on boiled eggs, as performed by Romanoff & Romanoff (1933). In some cases, 3–5 volumes of distilled water were added to the non-incubated egg albumen. The resulting precipitate (ovomucin) was centrifuged down and discarded and the supernatant used for electrophoretic analysis.

Amniotic fluid was obtained by puncturing the amniotic sac, taking care to avoid any loss or contamination with the other embryonic fluids. Globulins were prepared as described for albumen.

Yolk was obtained by puncturing the vitelline membrane or the yolk sac and the WSF was prepared as previously described (Carinci et al. 1966). To prevent contamination with egg albumen, for the non-incubated eggs, yolks were first washed with tap water and carefully rolled on filter paper.

For each fluid (albumen, amniotic fluid and yolk) and for each stage of incubation we performed at least 10 independent determinations, whose arith-metic averages are recorded on the graphs.

The albumen and amniotic fluid dry weights were deter-mined gravimetrically on samples dried at 110 °C to constant weight; albumen values were corrected for added NaCL

The total and protein N content were estimated by the micro-Kjeldhal pro-cedure. To determine the protein N content, trichloroacetic acid (15%) was added to aliquots of the different fluids to give 5 % final concentration. The proteins were then centrifuged, washed with 10 % trichloroacetic acid and analyzed for their N content (protein = N × 6·25).

Samples, diluted with 0·16 M-NaCi to 2·5 % protein concentration, were examined on cellulose polyacetate (Gelmann Sepraphore III, 6 3/4 in. strips) in tris barbital-sodium barbital buffer, pH 8·8, ionic strength μ — 0·05, 300 V, 60 min. The strips were stained with amido schwartz. The individual proteins on electropherograms have been recognized both by their behaviour during electrophoresis and by comparison with isolated proteins from albumen (globulins and ovomucoid). For quantitative estimations strips were scanned with a Joyce-Loebl Chromoscan densitometer.

Samples were exhaustively dialysed against 1 M-NaCl at 4 °C, diluted to 1 % protein concentration and then examined in a Phywe model U50L analytical ultracentrifuge at 20 °C and 167241g. Calculated sedi-mentation coefficients (in Svedberg units S), referred to 1 % protein concentration, were corrected for temperature, viscosity and partial specific volume.

The albumen wet weight after 10 days of incubation is 30·3 % of that of the non-incubated egg; it then does not change until the 12th day of incubation, and thereafter diminishes steadily. After 17 days of incubation albumen is still present, in low amounts, in about 10 % of the embryos examined (Text-fig. 1 A).

The albumen dry weight remains nearly unchanged up to 12 days of incuba-tion, then decreases similarly to that of the wet weight (Text-fig. 1B).

The decrease in the residual albumen wet weight in the early stages of develop-ment is due mainly to water absorption (Needham, 1931 ; Romanoff, 1960). The ratio between dry and wet weight is 37 % after 10 days of incubation, compared with 11 % in the non-incubated egg.

Our quantitative values show moderate variations in embryos at the same developmental stages; these become pronounced after 14–16 days of incubation, at the same time as albumen absorption is taking place.

The total N content is nearly constant up to 12 days of incubation, and then diminishes like the dry weight (Text-fig. 1C). As proved by determinations on TCA precipitates, almost all the N content is present as protein N (Text-fig. 1D). The % content of N and proteins, calculated on a dry weight basis, does not show any noticeable variation during the incubation period studied.

The electrophoretic pattern of the non-incubated egg albumen, diluted with 0·16 M-NaCl, shows six zones with anodic mobility and one variable zone with cathodic mobility. The electrophoretic zones are identi-fiable by their position after migration and by comparison with isolated fractions as ovalbumin, which is divided into three components, globulins G3 and G2, conalbumin and lysozyme (Plate 1 a). The subdivision of ovalbumin into three bands is due to the fact that the phosphorus content is different in each fraction (Fevold, 1951). Sometimes ovalbumin divides into five bands with a better resolution than that observed on starch gel electrophoresis (Steven, 1961). By electrophoresis we did not succeed in separating zones of ovomucoid and ovomucin, proteins which have been isolated from non-incubated egg albumen (Warner, 1954).

The qualitative electrophoretic pattern of residual albumen after 10, 12, 13, 14, 15 and 16 days of incubation is the same as that of the non-incubated egg.

However during development there appears, on electropherograms, a zone of ovomucoid, identified by comparison with isolated ovomucoid (Plate 1 b, c).

The relative proportion of protein fractions on electrophoresis is given in Table I. For the non-incubated egg our values differ from those obtained by paper and free-boundary electrophoresis (Fevold, 1951; Rizzoli, 1956). In oui-opinion the presence of ovomucin in our electropherograms perhaps accounts for these differences; the densitométrie values of electropherograms of albumen diluted with H2O (causing ovomucin precipitation) agree with those already published (Table 1, column 2).

The relative proportion of the various fractions is constant during incubation.

The ultracentrifugal analysis of non-incubated egg albumen shows only one peak, with sedimentation coefficient of 2·6 S (Plate 2d). Only one peak is present during the entire development period. Sedimentation coefficients are given in Table 2.

Amniotic fluid volume is approximately 3 ml after 10 days of incubation; it increases up to 15 days, then decreases slowly up to 17 days and drops quickly on the 18th day, as previously reported (Romanoff, 1967) (Text-fig. 2A).

Amniotic fluid dry weight is very low at 12 days of incubation; it undergoes a quick increase after 14 days, and then it decreases, at first slowly and then at the same speed as the reduction in volume (Text-fig. 2B).

After 14 days of incubation about 90 % of the dry weight is protein, as shown by N analysis of TCA precipitates (Text-fig. 2C).

On the 14th day, seven anodic fractions and one irregular fraction migrating to the cathode can be observed on electrophero-grams; the qualitative pattern is very similar to that of albumen. The electro-phoretic pattern remains unchanged during the whole period examined (Plate 1 d, e).

The relative proportion of the different fractions is given in Table 1 ; it is nearly the same as that for albumen.

After 15 days of incubation the amniotic fluid in tolo shows only one peak on ultracentrifugal examination, even after 90 min centrifugation (Plate 2c); the sedimentation rate (2·6 S) agrees with that of the total albumen ultracentrifugal component. Only one peak can be found up to 17 days of incubation (Table 2).

At the 15th and the 17th day of incubation the globulin shows two peaks after 40 min centrifugation (Plate 2h, i). Their sedimentation rates are given in Table 2.

As previously reported, electrophoretic examination of WSF shows three bands (α, β γ and y-livetin) up to 13 days of incubation. Sometimes γ-livetin is divided into two bands (γ1 and γ2-livetin) (Mok & Common, 1964). Besides an ovalbumin migration fraction is present (Plate 1f) (Marshall & Deutsch, 1951). After 14 days the electrophoretic pattern undergoes some changes due to the appearance of the albumen proteins (Plate 1g, h). We have to call attention to the fact that, under our experimental conditions, γ1-livetin has a mobility slightly greater than that of conalbumin.

The relative proportion of the different fractions is given in Table 3. In non-incubated eggs β-livetin is the prevalent fraction; the ovalbumin component amounts to 2·5 %. The relative proportion of the various components remains unchanged up to 13 days of incubation. After 14 days ovalbumin is somewhat less than 20 % of total protein content, after 15 days it increases to 58·5 %. The increase in proportion of ovalbumin takes place mostly from 14 to 15 days of incubation. After 15 days, albumen proteins represent at least 65% of total protein content.

As a result of this study we have first to underline the fact that albumen solids absorption does not take place, in noticeable amounts, before 12 days of incubation. Indeed during this period dry weight and protein content are nearly the same as those of the non-incubated egg. Nevertheless, it is possible that during this period small quantities of albumen are absorbed. Electron microscopic evidence indicates the presence of albumen in blastoderm ectodermal cells (Ruggeri, 1967); moreover, proteins with the immunological behaviour of ovalbumin and conalbumin have been found in embryonic fluids at 5–8 days of incubation (Kaminski & Durieux, 1954; Kaminski & Durieux, 1956).

Albumen is therefore a reserve protein material which the embryo does not utilize, in noticeable amounts, before the second incubation phase. In early development nourishment for the embryo is provided almost completely from yolk proteins (Rupe & Farmer, 1955; Walter & Mahler, 1958).

The residual albumen protein composition remains substantially unchanged during incubation and hence is very similar to that of the non-incubated egg. This agrees with previously reported electrophoretic data. All the fractions are absorbed in the same relative proportion, so we can exclude any preferential absorption of ovalbumin.

After 13 days of incubation albumen absorption begins. The absorption becomes very high after 14 days; at this time a 5-fold increase in amniotic fluid protein content is observable. Amniotic fluid proteins are identifiable as albumen proteins by their electrophoretic and ultracentrifugal behaviour. Even the rela-tive proportion of the different fractions is very similar to that of albumen, and therefore it is clear that albumen passes unchanged into the amniotic sac.

After 14 days of incubation amniotic fluid proteins account for70 % of ab-sorbed albumen proteins. On the following day the amniotic fluid protein content increases; it is impossible however to quantify the absorbed albumen, because it is very likely that at the same time some proteins are absorbed from amniotic fluid by the embryo.

After 14 days of incubation albumen proteins are also present in the yolk.

In this research we have not determined their total amounts, mainly because all the proteins absorbed into yolk are not present in the WSF, a part being adsorbed on vitelline granules (as proved for ovalbumin by Martin & Saito (1967). At least 60% of WSF proteins, prepared according to our procedure, consists of albumen proteins. Taking into account the quantitative determina-tion by Saito et al. (1965) on WSF, this explains the absorption of about 530 mg of albumen proteins after 15 days of incubation. Albumen proteins are recovered from the yolk in a relative proportion very similar to that of albumen, as shown by densitométrie measurements on electropherograms. We have not observed reduction in ovalbuminx as compared with ovalbumin2, as found by Saito & Martin (1966).

The albumen absorption mechanism is under further study in our laboratory. As for the fate of albumen absorbed into yolk and amniotic sac we can formulate the following hypotheses :

The albumen proteins absorbed into yolk are likely to be hydrolyzed by yolk enzymes. Acid-soluble N, mainly due to amino acids, is present in very low concentration in albumen and amniotic fluid during all the period examined and in the yolk up to 13 days of incubation, and after 14 days increases remarkably in the yolk, just when albumen is absorbed (Mclndoe, 1960).

During the last incubation period embryonic protein synthesis becomes very great, as shown indirectly by conspicuous embryo growth and directly by the synthesis of specific proteins such as muscle proteins (Csapo & Herrmann, 1951 ; Herrmann, White & Cooper, 1957; Carinci & Manzoli, 1964). This synthesis utilizes amino acids and can be prevented by amino acid analogues (Wadding-ton & Perry, 1958; Carinci, Manzoli & Zaniboni, 1964). These increasing needs are likely to be met mainly through a supply of amino acids from albumen proteins hydrolyzed in the yolk.

It is more difficult to explain the significance of albumen absorption into the amniotic sac. It is known that some amniotic fluid is ingested by the embryo (stained particles introduced into amniotic sac have been found in the alimentary channel (Hamilton, 1952). In this way albumen proteins are supplied to the developing embryo. Secondly, the increase of amniotic protein content would probably cause a rise in viscosity which could be related to the protective func-tion of this fluid.

1. Study of albumen absorption during chick embryogenesis was carried out, determining wet weight, dry weight and protein content of residual albumen and amniotic fluid, and the relative proportion of yolk water-soluble fraction proteins.

2. Electrophoretic and ultracentrifugal analysis of proteins of these embryo-nic fluids have been carried out.

3. In early development albumen wet weight decreases sharply, while dry weight and protein content remain nearly unchanged up to 12 days of incubation and very similar to those of non-incubated egg. They then decrease slowly up to 14 days and more quickly up to 17 days of incubation.

4. During development the qualitative composition and the relative pro-portion of the residual albumen proteins remain nearly unchanged as judged by electrophoretic and ultracentrifugal examination.

5. After 13 days of incubation protein content of amniotic fluid increases rapidly; the recovered proteins are identifiable as albumen proteins by electro-phoretic and ultracentrifugal examination.

6. After 14 days of incubation albumen proteins are found in the yolk in relative proportions very similar to those of albumen.

7. Almost all of the albumen is absorbed across yolk and amnion. The embryological significance of these processes is discussed.

1. E’ stato esaminato il riassorbimento dell’albume durante lo sviluppo dell’embrione di pollo mediante valutazione del peso fresco, peso secco e con-tenuto proteico dell’albume e del fluido amniotico ed inoltre vaiutando la concentrazione relativa delle component! proteiche della frazione idrosolubile del vitello.

2. Si è anche eseguita l’analisi qualitativa e quantitativa mediante ultracentri-fugazione ed elettroforesi delle proteine di tali fluidi embrionali.

3. Mentre il peso fresco dell’albume diminuisce fortemente nel primo periodo di incubazione, peso secco e contenuto proteico rimangono pressochè immodifi-cati fino al 12° g, per diminuiré lentamente fino al 14° g e rápidamente fino al 17° g di incubazione.

4. Durante lo sviluppo la composizione qualitativa e la concentrazione rela-tiva delle proteine dell’albume residuo rimangono pressochè immodificate e dello stesso ordine dell’uovo non incubato (analisi all’elettroforesi e all’ultra-centrifuga).

5. Dopo il 13° g il contenuto proteico del fluido amniotico aumenta forte-mente; le proteine ivi ritrovabili sono identificabili corne proteine dell’albume (analisi all’elettroforesi e all’ultracentrifuga).

6. Dal 14° g si ritrovano nel vitello e precisamente nella frazione idrosolubile proteine dell’albume in concentrazione relativa analoga a quella con cui si ritrovano nell’albume.

7. L’albumeèpressochè totalmente riassorbito via vitello e fluido amniotico; viene discusso il signifícate embriológico di tale fatto.