Immunochemical Analysis of the Water-Soluble Fraction of the Chick Embryo Yolk

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J. Embryo/, exp. Morph. Vol. 24, 1, pp. 13-20, 1970 13 Printed in Great Britain Immunochemical analysis of the water-soluble fraction of the chick embryo yolk

By C. E. GROSS!,1 P. CARINCP AND L. MANZOLI-GUIDOTTF From the Institute of Anatomy, University of Genoa and the Institute of Histology and General Embryology, University of Bologna

SUMMARY By means of immunochemical techniques, the protein components of the water-soluble fraction (WSF) of the egg yolk have been examined in the unincubated egg and during incubation. Anti-ovalbumin and anti-total adult chicken serum antisera have been employed. Ovalbumin can be detected in the unincubated WSF as well as during incubation; its concentration seems to increase during incubation. In the WSF of the unincubated egg, six proteins immunologically related to adult serum proteins can be detected. They correspond to a-livetin, o^-globulin, /?-livetin, ovotransferrin (conalbumin) and y-livetin (two components). /?-Livetin disappears after the 14th day of incubation while the other components can be demonstrated till hatching. The findings are discussed in relation to the data available in the literature.

INTRODUCTION The water-soluble fraction (WSF) of the unincubated hen's egg yolk (livetin fraction) (Plimmer, 1908) shows, in moving boundary electrophoresis, three main components (a-, /?-, and y-livetin) (Shepard & Bottle, 1949; Martin, Vandegaer & Cook, 1957); by paper electrophoresis the y-livetin zone can be partially re- solved into two bands (yx- and y2-livetin) (Knight & Schechtman, 1954; Mok & Common, 1964), while, by the use of cellulose polyacetate, a fourth minor component with an ovalbumin migration pattern can be detected (Carinci & Manzoli-Guidotti, 1968). Immunoelectrophoretic investigations have demonstrated the immunological identity of the major protein components of the yolk WSF with serum proteins (Williams, 1962; Mok & Common, 1964; Zaccheo & Grossi, 1967); namely, a-livetin corresponds to serum albumin, /?-livetin to serum a2-globulin and y- livetin to serum y-globulin. The identification of the electrophoretic yj-livetin fraction with serum components is still under discussion (Mok & Common, 1964). In addition, ovotransferrin (conalbumin) and o^-globulin can be detected 1 Author's address: Institute of Anatomy, University of Genoa, Genoa, Italy. 2 Author's address: Department of Histology and General Embryology, Faculty of Medi- cine and Surgery, University of Perugia, Perugia, Italy. 3 Author's address: Institute of Histology and General Embryology, University of Bologna, Bologna, Italy. 14 C. E. GROSSI AND OTHERS in the WSF by means of immunological techniques, while the presence of ovalbumin is still uncertain (Marshall &Deutsch, 1951; Williams, 1962; Stratil, 1967). During incubation the protein composition of the yolk WSF undergoes some changes, as shown by electrophoretic and ultracentrifugal analysis; some components disappear while the yolk is enriched by new proteins absorbed from the egg white (Saito, Martin & Cook, 1965; Saito & Martin, 1966; Carinci, Wegelin & Manzoli-Guidotti, 1966; Carinci & Manzoli-Guidotti, 1968). The data in the literature do not show a full agreement about such modifica- tions; therefore we have analysed the protein composition of WSF in various developmental stages by means of immunochemical methods employing anti- total chicken serum and anti-ovalbumin antisera.

MATERIAL AND METHODS We have used White Leghorn fertile eggs, provided by the Corticella Agri- cultural Station (Bologna). The WSF was prepared from unincubated eggs and after 12, 14, 15 and 21 days of incubation (38 °C, 60% relative humidity), according to Martin et al. (1957), as previously described (Carinci, Wegelin & Manzoli-Guidotti, 1966). Globulins were prepared by repeated precipitations with ammonium sulphate (Carinci & Manzoli-Guidotti, 1968). The anti-total chicken serum antisera (anti-TACS) have been supplied by Behringwerke. Anti-ovalbumin antisera have been prepared in our laboratories by the use of ovalbumin extracted from unincubated eggs and purified according to Warner's technique (Warner, 1954). Rabbit antisera against ovalbumin have been obtained following different immunization schedules. The first group of rabbits received subcutaneously 40 mg of antigen in a volume of 2 ml together with 2 ml of complete Freund's adjuvant. Repeat injections (10 mg of antigen each) were given intravenously and animals were bled when their serum reached a precipitation titre of about 1/32000 (a-precipitation test). The second group of rabbits received 20 mg of ovalbumin intraperitoneally and a series of subcutaneous repeat injections (10 mg of antigen each) was given until the same antibody titre was obtained. Anti-TACS and anti-ovalbumin antisera have been tested against the antigens by the immunoelectrophoretic microtechnique of Scheidegger (1955) and the Ouchterlony (1964) double gel immunodiffusion. The two antisera were always used at a constant litre, while the antigen solutions were adjusted to the same nitrogen concentration (micro-Kjeldahl).

RESULTS The purified ovalbumin fraction shows, on immunoelectrophoretic analysis against the homologous antiserum, only one precipitation line in the ovalbumin zone (Fig. 1A). Therefore it contains only one antigenic component and it behaves like a highly purified preparation. Immunochemical analysis of yolk 15 The anti-ovalbumin antiserum gives a single arc in the ovalbumin zone when tested against WSF prepared from unincubated eggs (WSF0) (Fig. IB). The same component can be detected in WSF after 12, 14, 15 and 21 days of incubation (e.g. Fig. 2 A, B). On the other hand, a slight change can be observed in the immunoelectrophoretic pattern after 15 and 21 days; the different shape of the precipitation arc is possibly related to a higher concentration of the antigen during the latter stages.

Fig. 1. Immunoelectrophoretic pattern of ovalbumin (A) and WSF0 (B) against anti-ovalbumin antiserum.

Ovalbumin can also be detected in WSF0, WSF12, WSF14, WSF15 and WSF21 by the double diffusion tests (Fig. 3). Immunoelectrophoretic investigations performed on adult chicken serum against anti-TACS antiserum show the presence of at least 15 antigenic components (Fig. 4A). The same antiserum when tested against WSF0 forms six precipitation lines whose electrophoretic migration rate corresponds respectively to albumin, ^-globulin, a2-globulin, ^-globulin and y-globulin (two overlapping components which might correspond to IgM and IgG: immunoglobulins M and G) (Fig. 4B). From earlier work (Williams, 1962; Mok & Common, 1964), the albumin-like fraction can be identified with a-livetin, the a2-globulin-like fraction with /Mivetin, and the y-globulin-like fraction with the faster cathodic migration rate can be identified with y-livetin. In addition, the /?-globulin-like fraction detected by us should correspond to ovotransferrin (conalbumin), 16 C. E. GROSSI AND OTHERS

A WSF12 O Anti-OvA I ) — ^ O

WSF1S B

Fig. 2. Immunoelectrophoretic pattern of anti-ovalbumin antiserum against WSF12 (A) and WSF15 (B).

o o o WSF0 OvA 0 , 0 0 o fr Anti-OvA VJ^y ft (C^ (<- 0 o • 0 0

WSF21 WSF1S WSF12 o OvA

Fig. 3. Ouchterlony test. The central holes contain anti-ovalbumin antiserum. In the peripheral holes are different preparations from WSF and ovalbumin. The holes labelled with dots contain other antigenic samples. Lmmunochemical analysis of yolk 17 while the y-globulin-like component with the slower cathodic migration rate might be identified with yi-livetin. 1 The same electrophoretic pattern shown by WSF0 can be observed with WSF12 (Fig. 5A) and WSF14. On the contrary, WSF15 when tested against anti-TACS antiserum shows a different pattern. As previously, a-livetin, ar ) globulin, ovotransferrin and the two -/-globulin precipitation lines can be detected, while the arc corresponding to /?-livetin has disappeared (Fig. 5B). The same finding can be obtained after 21 days of incubation (Fig. 6A).

Fig. 4. Immunoelectrophoretic patterns given by anti-TACS antiserum against TACS (A) and WSF0 (B).

These observations are confirmed by the Ouchterlony tests. The ammonium sulphate precipitates prepared from WSF0 (WSFP0), when tested against anti-TACS antiserum, give three precipitation lines; one is found in the a2-globulin zone and two in the y-globulin area. The latter might be respectively referred to as IgM and IgG. Two lines in the y-globulin zone can also be observed in WSFP15 and WSFP21.

DISCUSSION The detection by immunological methods of ovalbumin in the yolk extracts prepared from unincubated eggs confirms previous electrophoretic observations (Carinci & Manzoli-Guidotti, 1968). This finding corresponds with observations by Marshall & Deutsch (1951) and by Stratil (1967), but not with those of Williams (1962). Such a disagreement could be explained by the fact that

2 EM B 24 18 C. E. GROSSI AND OTHERS I

Fig. 5. A comparison of the immunoelectrophoretic patterns given by WSF12 \ (A) and WSF15 (B) against anti-TACS antiserum. In the latter is shown the dis- appearance of /?-livetin (arrow).

Fig. 6. A, Immunoelectrophoretic pattern given by WSF21 against anti-TACS antiserum. The yff-livetin arc is lacking. B, Globulin components in a fraction prepared from albumen; they are antigenically related to WSF and serum globulins. Immunochemical analysis of yolk 19 the last observation was made only by double immunodiffusion which, especially in case of complex antigens like WSF, may be faulty because of the masking of some components. WSF0 contains, in addition, other proteins which give an immunological re- action identical with that of albumen proteins, i.e. ovotransferrin (conalbumin) I and two globulin fractions, as shown by testing the same anti-TACS antiserum against WSF and albumen (Fig. 6A, B). It seems appropriate to stress that ovalbumin can also be detected in the subgerminal fluid (unpublished data), which represents an early nutrient of the developing embryo (Elias, 1964). The ovalbumin content of WSF seems to undergo a progressive increase during incubation, as judged from the evolution of the immunoelectrophoretic pattern and according to previous quantitative evaluations (Saito et al. 1965; Carinci et al 1966; Carinci & Manzoli-Guidotti, 1968). Six proteins immunologically identical with adult homologous serum proteins have now been detected in yolk WSF0. Williams (1962) has found only four of these and does not record arglobulin or the first y-globulin component. Mok & Common (1964) have been able to show the o^-globulin fraction. These different findings can be referred to the different antibody pattern of the tested anti-TACS antisera; our anti-TACS antiserum has a very wide antibody range and therefore it is able to detect a higher number of antigenic components. The paper electrophoretic y^livetin fraction should correspond to ovotransferrin, according to Hui & Common (1966); in our opinion it is possible that, at least partly, this fraction contains LgM (macroglobulin), even though we have not been able to demonstrate any macroglobulin in WSF before the 14th day of incubation by means of ultracentrifugal analysis (Carinci & Man- zoli-Guidotti, 1968). On the basis of immunological observations we suggest that a macroglobulin is already detectable in the yolk of unincubated eggs. The immunoelectrophoretic analysis of the protein pattern in WSF during incubation has shown that /Mivetin disappears while the other components are all detectable till hatching. On the basis of electrophoretic observations Saito & Martin (1966) conclude that a-livetin disappears from the WSF after the 15th day of incubation; on the other hand Carinci & Manzoli-Guidotti (1968) have been able to demonstrate this protein while /?-livetin seemed to disappear after the 15th day. The present data confirm the observations of the latter authors. In conclusion, by means of the immunological analysis it has been shown that (1) ovalbumin is present in the yolk before and during incubation and (2) /Mivetin disappears from the yolk after the 14th day of incubation, but the other components remain. 20 C. E. GROSSI AND OTHERS

RIASSUNTO Anal isi immunochimica della frazione idrosolubile del vitello dell' embrione di polio. Per mezzo di tecniche immunochimiche e stata studiata la composizione proteica della frazione idrosolubile del vitello dell'uovo di polio non incubato e durante lo sviluppo dell'embrione. La frazione idrosolubile e stata esaminata in toto e nelle sue componenti globuliniche, con l'impiego di sieri anti-ovalbumina e anti-sieroproteine totali di polio adulto. Ovalbumina e immunologicamente dimostrabile nell'uovo non incubato e per tutto il periodo dello sviluppo; la sua concentrazione sembra aumentare durante l'incubazione. Nella frazione idrosolubile dell'uovo non incubato si ritrovano sei proteine immunologicamente identiche a proteine del siero adulto; esse corrispondono ad a-livetina, o^-globulina, /?-livetina, ovotransferrina (conalbumina), y-livetina (due componenti). Durante l'incubazione si osserva la scomparsa della /?-livetina dopo il 14° giorno mentre le altre componenti persistono fino alia schiusa. II significato di questi reperti viene esaminato in relazione con i dati della letteratura.

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(Manuscript received 6 August 1969)