/ . Embryol. exp. Morph. Vol. 20, 1, pp. 107-18, August 1968 107With 2 plates

Printed in Great Britain

Albumen absorption during chick embryogenesis

By P. CARINCI1 & L. MANZOLI-GUIDOTTI1

Institute of Histology and General Embryology, University of Bologna

In birds albumen represents a substantial part of the reserve material for theirembryological development (Romanoff & Romanoff, 1949). In Gallus gallusalbumen proteins form 50 % of non-incubated egg protein content (Romanoff &Romanoff, 1949). During incubation such material is absorbed and utilized, toa great extent, for the synthesis of embryonal proteins (Fiske & Boyden, 1926;Rupe & Farmer, 1955). It is not yet well understood how these processes (albumenabsorption and utilization for the energetic and nutritional needs of the embryo)take place. The present available data indicate that the amount of albumen solidsbegins to diminish starting from 11 to 12 days of incubation (Romanoff &Romanoff, 1933; Rupe & Farmer, 1955). From 13 to 14 days, after the sero-amniotic connexion perforates, the protein content of the amniotic fluid showsa marked increase because of the passage of albumen (Needham, 1931; Romanoff,1960). During the same development period albumen proteins can be recovered,in large amounts, in the yolk (Saito, Martin & Cook, 1965; Carinci, Wegelin &Manzoli-Guidotti, 1966).

Indeed the major part of albumen is very likely absorbed across the yolk andthe amnion. In this connexion the function of the albumen sac is still not clearlyknown. This sac, formed starting from the 9th to 10th day of incubation by afolding of the chorioallantois, is considered by some authors to be responsiblefor albumen absorption (Patten, 1950).

During development, the protein composition of the residual albumen remainssubstantially unchanged and very similar to that of the non-incubated egg (asjudged by paper and free-boundary electrophoresis) (Marshall & Deutsch, 1950;Rizzoli, 1956); besides, the immunological behaviour of the individual proteinsis unmodified (Kaminski & Durieux, 1954). The electrophoretic data indicatealso that the relative proportion of different albumen proteins is constant duringincubation (Marshall & Deutsch, 1950), though some quantitative observationsdemonstrate 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).

1 Authors' address: Istituto di Istologia e Embriologia Generale, Universita di Bologna, ViaBelmeloro, Bologna, Italy.

108 P. CARINCI & L. MANZOLI-GUIDOTTI

With regard to the yolk it has been demonstrated that the albumen proteinscan be recovered from the water soluble fraction (WSF) (Carinci & Manzoli-Guidotti, 1968).

Up to the present time, however, there is no analysis performed simultaneouslyon embryonic fluids to furnish a complete picture of the qualitative and quanti-tative modes of albumen absorption. In this research we have followed thealbumen absorption, determining at the same time the wet weight, dry weightand protein content of residual albumen and amniotic fluid, and also estimatingrelative proportion of yolk WSF proteins. In addition we have carried outelectrophoretic and ultracentrifugal analysis of the proteins of these embryonicfluids.

MATERIALS AND METHODS

We have used eggs (60 ± 0-5 g, average weight) laid by a small group of WhiteLeghorn hens in June-October 1967, provided by the Corticella agriculturalstation (Bologna). The eggs were incubated at 38±0-5°C, at 60% relativehumidity and turned twice a day.

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

Albumen was removed in toto, including the chalazae, immediately weighedand diluted with 0-16 M NaCl (1:2, w/w) to obtain an albumen dilution withoutovomucin precipitation (Forsythe & Foster, 1950); then the albumen was homo-genized with a Waring blender. From this material globulins were prepared byrepeated 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 isolatingalbumen from yolk sac, quantitative determinations were also carried out onboiled 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 andthe supernatant used for electrophoretic analysis.

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

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

For each fluid (albumen, amniotic fluid and yolk) and for each stage of

Albumen absorption 109

incubation we performed at least 10 independent determinations, whose arith-metic averages are recorded on the graphs.

Chemical analyses. The albumen and amniotic fluid dry weights were deter-mined gravimetrically on samples dried at 110 °C to constant weight; albumenvalues 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 %) wasadded to aliquots of the different fluids to give 5 % final concentration. Theproteins were then centrifuged, washed with 10 % trichloroacetic acid andanalyzed for their N content (protein = N x 6-25).

Electrophoretic analyses. Samples, diluted with 0-16 M-NaCl to 2-5 % proteinconcentration, were examined on cellulose polyacetate (Gelmann SepraphoreIII, 1 x 6f in. strips) in tris barbital-sodium barbital buffer, pH 8-8, ionicstrength /i = 0-05, 300 V, 60 min. The strips were stained with amido Schwartz.The individual proteins on electropherograms have been recognized both by theirbehaviour during electrophoresis and by comparison with isolated proteins fromalbumen (globulins and ovomucoid). For quantitative estimations strips werescanned with a Joyce-Loebl Chromoscan densitometer.

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

RESULTS

1. Albumen

Quantitative analyses. The albumen wet weight after 10 days of incubation is30-3 % of that of the non-incubated egg; it then does not change until the 12thday of incubation, and thereafter diminishes steadily. After 17 days of incubationalbumen is still present, in low amounts, in about 10 % of the embryos examined(Text-fig. 1A).

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). Theratio between dry and wet weight is 37 % after 10 days of incubation, comparedwith 11 % in the non-incubated egg.

Our quantitative values show moderate variations in embryos at the samedevelopmental 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 thendiminishes like the dry weight (Text-fig. 1C). As proved by determinations on

110 P. CARINCI & L. MANZOLI-GUIDOTTI

TCA precipitates, almost all the Ncontent is present as protein N (Text-fig. 1D).The % content of N and proteins, calculated on a dry weight basis, does notshow any noticeable variation during the incubation period studied.

40 -

30 -

| 20

! 10 -

TT 4 -

>* 3 -

2 -

1 -

0-6'So ,. _~ 0-5

| 0-4

J 0-3| 02

0-1

4 h

3

2

1 -

•ss

s

\N

S.

1 / / I I I

1 / ( I I I

— . v

1 / / I I I

0 " 10 11 12

\

i i i • —

13 14 15 16

A

B

c

i—0

D

17

Incubation (days)

Text-fig. 1. Average values of albumen wet weight (A), dry weight (B),N (C) and protein content (D) for the indicated days of incubation.

Electrophoretic analyses. The electrophoretic pattern of the non-incubated eggalbumen, diluted with 0-16 M-NaCl, shows six zones with anodic mobility andone variable zone with cathodic mobility. The electrophoretic zones are identi-fiable by their position after migration and by comparison with isolated fractionsas ovalbumin, which is divided into three components, globulins G3 and G2,conalbumin and lysozyme (Plate 1 a). The subdivision of ovalbumin into threebands is due to the fact that the phosphorus content is different in each fraction(Fevold, 1951). Sometimes ovalbumin divides into five bands with a betterresolution than that observed on starch gel electrophoresis (Steven, 1961). By

S

1015

1417

1421

•§.

am-

Ele

ctro

phor

etic

pat

tern

s of

alb

umen

(a,

b,

c), a

mni

otic

flu

id {

d, e

), a

nd y

olk

wat

er-s

olub

le f

ract

ion

(/,

g, h

) fo

r th

e in

dica

ted

days

of

incu

batio

n. S

ee te

xt f

or e

xper

imen

tal

deta

ils.

o

> H W

/. Embryol. exp. Morph., Vol. 20, Part 1 PLATE 2

A

a-0 b-10 c-15

V

d-15 e-17b *

f-o

g-15 h-15 i-17

Ultracentrifugal patterns of albumen in toto (a, b, c), amniotic fluid in toto (d, e), albumenglobulins (/; g), and amniotic fluid globulins (/?, /) for the indicated days of incubation. Seetext for experimental details.

P. CARINCI & L. MANZOLI-GUIDOTTI

Albumen absorption 111electrophoresis we did not succeed in separating zones of ovomucoid andovomucin, 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.

Table 1. Relative proportion (%) of protein fractions

Fra

IIIIIIIVVVIVII

ctionsf

(OvO(Ova)(Ov.)(Om)(G.)(G,M(Ca)J

i

34-218-54 0—

13-3290

Albumen*(Days of incubation)

0

41-6J17-44-5—9-6

26-9

5

31-916-34-56-4

13126-8

10

36-215-53-46 1

13-225-6

14

37-116-85-64 0

10-925-6

15

36-515-93-44-8

12-6f 8-6118-2

Amniotic fluid(Days

14

32-918-5

3-86 0

12-55-8|

20-5J

of incubation)

15

33-617-54-16-5

10-2281

16

35-614-94-45-8

14-2251

VIII (Li) 10 — i-o — — — — — —

* Diluted with 016 M-NaCl.t Numbered starting from anode (top) to cathode (bottom). In the parentheses the principal

protein component is indicated.% Diluted with 3/5 volumes of water.

Table 2. Sedimentation

Total albumenS

Precipitated albumen*(fl)S(b)S

Total amniotic fluidS

Precipitated amniotic fluid(a)S(b)S

coefficients

0

2-6

3116-3

—

——

of protein components

Days of incubation

10

2-5

3 015-7

—

——

15

2-7

3-316-2

2-6

3-0151

17

—

——

2-6

2-8160

S = Svedbergs at 20 °C and 1 % protein concentration, (a) and (b) indicate theindividual peaks of various fractions.

* Previously published (Carinci & Manzoli-Guidotti, 1968).

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

The relative proportion of protein fractions on electrophoresis is given inTable 1. For the non-incubated egg our values differ from those obtained by

112 P. CARINCI & L. MANZOLI-GUIDOTTI

paper and free-boundary electrophoresis (Fevold, 1951; Rizzoli, 1956). In ouropinion the presence of ovomucin in our electropherograms perhaps accountsfor these differences; the densitometric values of electropherograms of albumendiluted with H2O (causing ovomucin precipitation) agree with those alreadypublished (Table 1, column 2).

The relative proportion of the various fractions is constant during incubation.Ultracentrifugal analyses. The ultracentrifugal analysis of non-incubated egg

albumen shows only one peak, with sedimentation coefficient of 2-6 S (Plate 2a).Only one peak is present during the entire development period. Sedimentationcoefficients are given in Table 2.

2. Amniotic fluid

Quantitative analyses. Amniotic fluid volume is approximately 3 ml after10 days of incubation; it increases up to 15 days, then decreases slowly up to17 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 undergoesa quick increase after 14 days, and then it decreases, at first slowly and then at thesame speed as the reduction in volume (Text-fig. 2B).

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

Electrophoretic analyses. On the 14th day, seven anodic fractions and oneirregular 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\d,e).

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

Ultracentrifugal analyses. After 15 days of incubation the amniotic fluidin toto shows only one peak on ultracentrifugal examination, even after 90 mincentrifugation (Plate 2 c); the sedimentation rate (2-6 S) agrees with that ofthe total albumen ultracentrifugal component. Only one peak can be found upto 17 days of incubation (Table 2).

At the 15th and the 17th day of incubation the globulin shows two peaks after40 min centrifugation (Plate 2h, /). Their sedimentation rates are given in Table 2.

3. Yolk water-soluble fraction

As previously reported, electrophoretic examination of WSF shows threebands (a-, /?- and y-livetin) up to 13 days of incubation. Sometimes y-livetin isdivided into two bands (yx- and y2-livetin) (Mok & Common, 1964). Besides anovalbumin migration fraction is present (Plate 1/) (Marshall & Deutsch, 1951).After 14 days the electrophoretic pattern undergoes some changes due to the

Albumen absorption 113

« ow UO <D

11 12 13 14 15 16 17 18Incubation (days)

Text-fig. 2. Average values of amniotic fluid volume (A), dry weight (B)and protein content (C) for the indicated days of incubation.

Table 3. Yolk WSF. Relative proportion (%) of protein fractions

Fractions*

IIIIIIIVVVI

(a-liv)(OvJ(Ov2)(Ov3)(A-liv, Om)(G3)

VII (G^- l i v )VIII(Ca,ya-liv)IX

* Numberec

(Li)

1 starting from

t

0

15-62-5——

470—

34-7}

—anode (top)

protein components are indicated.

Days

13

15-42-8——

43-2—

38-6J

—to cathode

of incubation

14

1101017-7—

32-4—

38-8}

—(bottom).

15

5-340-214-24-13-87-6

23-2J-10

21

3-438-7101417-0

110

25-7J

—In parentheses the principal

J E E M 20

114 P. CARINCI & L. MANZOLI-GUIDOTTI

appearance of the albumen proteins (Plate \g, h). We have to call attention tothe fact that, under our experimental conditions, yj-livetin has a mobility slightlygreater than that of conalbumin.

The relative proportion of the different fractions is given in Table 3. In non-incubated eggs /?-livetin is the prevalent fraction; the ovalbumin componentamounts to 2-5 %. The relative proportion of the various components remainsunchanged up to 13 days of incubation. After 14 days ovalbumin is somewhatless than 20 % of total protein content, after 15 days it increases to 58-5 %. Theincrease in proportion of ovalbumin takes place mostly from 14 to 15 days ofincubation. After 15 days, albumen proteins represent at least 65% of totalprotein content.

DISCUSSION

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

Albumen is therefore a reserve protein material which the embryo does notutilize, in noticeable amounts, before the second incubation phase. In earlydevelopment nourishment for the embryo is provided almost completely fromyolk proteins (Rupe & Farmer, 1955; Walter & Mahler, 1958).

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

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

After 14 days of incubation amniotic fluid proteins account for7 0 % of ab-sorbed albumen proteins. On the following day the amniotic fluid protein contentincreases; it is impossible however to quantify the absorbed albumen, becauseit is very likely that at the same time some proteins are absorbed from amnioticfluid by the embryo.

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

Albumen absorption 115In this research we have not determined their total amounts, mainly because allthe proteins absorbed into yolk are not present in the WSF, a part beingadsorbed 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 mgof albumen proteins after 15 days of incubation. Albumen proteins are recoveredfrom the yolk in a relative proportion very similar to that of albumen, as shownby densitometric measurements on electropherograms. We have not observedreduction in ovalbumin! 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 formulatethe following hypotheses:

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

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

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

SUMMARY

1. Study of albumen absorption during chick embryogenesis was carried out,determining wet weight, dry weight and protein content of residual albumen andamniotic 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 dryweight and protein content remain nearly unchanged up to 12 days of incubation

8-2

116 P. CARINCI & L. MANZOLI-GUIDOTTI

and very similar to those of non-incubated egg. They then decrease slowly up to14 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 byelectrophoretic and ultracentrifugal examination.

5. After 13 days of incubation protein content of amniotic fluid increasesrapidly; 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 inrelative proportions very similar to those of albumen.

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

RIASSUNTO

Riassorbimento deWalbume durante lo sviluppo delVembrione di polio

1. E' stato esaminato il riassorbimento dell'albume durante lo sviluppodeU'embrione di polio mediante valutazione del peso fresco, peso secco e con-tenuto proteico dell'albume e del fluido amniotico ed inoltre valutando laconcentrazione relativa delle componenti proteiche della frazione idrosolubiledel vitello.

2. Si e 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 periododi incubazione, peso secco e contenuto proteico rimangono pressoche immodifi-cati fino al 12° g, per diminuire lentamente fino al 14° g e rapidamente fino al17° g di incubazione.

4. Durante lo sviluppo la composizione qualitativa e la concentrazione rela-tiva delle proteine dell'albume residuo rimangono pressoche immodificate edello 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 come proteine dell'albume{analisi all'elettroforesi e aH'ultracentrifuga).

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

7. L'albume e pressoche totalmente riassorbito via vitello e fluido amniotico;viene discusso il significato embriologico di tale fatto.

Albumen absorption 117

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(Manuscript received 8 January 1968)