/ . Embryo!, exp. Morph. Vol. 21, 3, pp. 467-84, June 1969 4 6 7Printed in Great Britain

Ultrastructural changes in the avian vitellinemembrane during embryonic development

By CYNTHIA JENSEN1

From the Department of Zoology, University of Minnesota, Minneapolis

The vitelline (yolk) membrane of the avian egg plays a dual role during earlyembryonic development; it encloses the yolk and provides a substratum forexpansion of the embryo (Fig. 1). Expansion appears to be dependent upon themovement of cells at the edge of the blastoderm which is intimately associatedwith the inner layer of the vitelline membrane (New, 1959; Bellairs, 1963).The blastoderm (embryonic plus extraembryonic cells) has almost coveredthe entire surface of the yolk by the third and fourth days of incubation, andwhen this stage has been reached the vitelline membrane ruptures over theembryo and slips toward the vegetal pole.

Embryonic axisPellucid area

Opaque area —/. jfej- .'• ; ' 0 ' . ^ $ ^ \ Shell

Yolk

Vitellinemembrane

Albumen

Fig. 1. Diagram of a longitudinal section through a chick egg incubated for 48 h.The body of the embryo is cut transversely.

Rupture of the membrane during development appears to be the consequenceof a decrease in its mechanical strength (Moran, 1936), which changes mostrapidly at the animal pole (over the embryo). After 48 h of incubation, the

1 Author's address: Department of Pathology, College of Medicine, University of Utah,Salt Lake City, Utah 84112, U.S.A.

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mechanical strength of the membrane is decreased by 73 % at the animal poleand only 37 % at the vegetal pole (Mesarosh, 1957).

The factors responsible for weakening of the membrane during developmenthave not been determined. Romanoff & Romanoff (1949) suggested that diffu-sion of water from the albumen into the yolk during incubation of eggs causesthe yolk to swell, stretching and weakening the vitelline membrane. Loss ofstrength may be due to the loss of a layer from the membrane (Fromm, 1964)or digestion of the membrane by a tryptic protease located in the albumen(Balls & Swenson, 1934). Spratt (1963) has suggested that the embryonic cellschange the nature of adjacent areas of the vitelline membrane with which theyare in close association. Since weakening of the membrane is greatest in the arealocated over the embryo, several investigators have suggested that the cells maysecrete an enzyme similar to the hatching proteases of invertebrates and lowervertebrates which digests the vitelline membrane (Moran, 1936; Ragozina,1957).

Bellairs, Harkness & Harkness (1963) suggested that during developmentstructural changes in the membrane accompany the decrease in mechanicalstrength. As they found no changes in thickness of the vitelline membraneduring incubation, they suggested that the decrease in mechanical strength is aresult of qualitative changes in the membrane rather than quantitative ones.Although histochemical changes are known to occur in the vitelline membraneduring early development (Shalumovich, 1960), no structural changes have beenreported. The present study indicates that there are progressive ultrastructuralchanges in the vitelline membrane during development which correlate withchanges in the strength of the membrane and may be produced through activitiesof the embryonic cells.

MATERIALS

Pieces of vitelline membrane which had been in contact with embryonic cellsfor varying lengths of time were selected for observation (refer to Fig. 1). Sincethe blastoderm expands radially during development, a piece of vitelline mem-brane located directly over the embryonic axis or pellucid area has been incontact with the cells of the embryo longer than a piece over the opaque area,the area vitellina. At early stages of development, a piece of vitelline membranefrom the vegetal pole has not yet been in contact with embryonic cells. The yolksac reaches the vegetal pole at approximately 96 h of incubation; therefore apiece of vitelline membrane taken from the vegetal pole after 106 h of incuba-tion has been in contact with embryonic cells for 10 h. Consequently, there aretwo ways to measure the amount of time the cells have been in contact with thevitelline membrane: (1) one may take pieces of vitelline membrane fromanalogous areas in eggs of different stages of incubation, or (2) one may takepieces of membrane from different parts of the same incubated egg. Vitellinemembranes from the following eggs were examined by electron microscopy:

Avian vitelline membrane 469

(i) Eight freshly laid chicken (White Leghorn) eggs; six were fertilized and twounfertilized.

(ii) Thirty-three fertilized chicken eggs incubated at 38 °C for periods rangingfrom 9 to 134 h.

(iii) Six freshly laid turkey (Broad-White-Nicholas) eggs; four were ferti-lized and two unfertilized.

(iv) Fourteen fertilized turkey eggs incubated at 38 °C for periods rangingfrom 48 to 186 h.

(v) Four freshly laid fertilized pigeon (Columba livid) eggs.(vi) Three fertilized pigeon eggs incubated at 38 °C for 10, 48 and 72 h.

METHODS

A. Electron microscopyThe egg shell was cracked, and the contents were poured into a fingerbowl

containing enough chick Ringer solution (7-19 g NaCl, 0-42 g KC1, 0-24 g CaCl2

in 1 1. distilled water) to cover the yolk. The embryos were staged (Hamburger &Hamilton, 1951; Mun & Kosin, 1960) and several areas of the vitelline mem-brane (usually from the animal pole, vegetal pole and over the peripheral edgeof the blastoderm) were cut out with scissors and transferred to a Petri dishcontaining fresh Ringer solution. Adhering yolk and albumen were quicklyremoved with forceps and the pieces of membrane were placed in the fixative.Several membranes were rinsed in sodium Veronal or 01 M phosphate buffersrather than Ringer solution. The majority of specimens were fixed for 1 h in1 % ice-cold osmium tetroxide buffered with sodium Veronal to a pH of 7-4(Palade, 1952). Some specimens were fixed for 30min in 4 % glutaraldehydebuffered to pH 7-4 with 0-1 M phosphate buffer (Sabatini, Bensch & Barnett,1963), followed by postfixation for 1 h in 1 % ice-cold osmium tetroxide bufferedto pH 7-4 with phosphate buffer. After fixation in the cold, most specimens wererapidly dehydrated in an acetone series, and embedded in Vestopal W (MartinJaeger, Geneva, Switzerland). Other material was dehydrated in an alcoholseries, and embedded in Araldite (Glauert & Glauert, 1958). Some specimenswere stained for 20 min to 1 h in 1 % phosphotungstic acid in absolute alcoholafter dehydration. Thin sections were cut on a Porter-Blum microtome withglass knives and picked up on formvar-covered grids which had been stabilizedby a light coating of evaporated carbon. Most sections were stained by floatingthe grids on the surface of a 2 % aqueous solution of uranyl acetate for 10-30min at room temperature. Others were stained with uranyl acetate followed bylead hydroxide for 20 min. All pictures were taken with an RCA EMU-3Delectron microscope operated at 50 KV.

AH electron micrographs in this paper are of material fixed in osmiumtetroxide, embedded in Vestopal W, and stained with uranyl acetate. Materialprepared in the other ways mentioned above did not appear to differ from thematerial presented here.

470 C. JENSEN

B. Surface replicas of the vitelline membrane

Satisfactory replicas were difficult to obtain. A method modified from Bradley(1960, p. 104) and presented in detail elsewhere (Jensen, 1966) gave the mostconsistent results. The two-stage method used involved making a primaryformvar impression of the fixed vitelline membrane and a secondary carbonreplica which was placed on a grid and examined in the electron microscope.

C. Culture of chick embryos on pieces of vitelline membraneThe methods used for culture of embryos on the surface of the vitelline

membrane are identical to those described by Spratt (1963). Ten-hour chickembryos were explanted with their ectodermal surface against the inner surfaceof vitelline membranes on a solid agar-chick extract medium and incubated forthe following lengths of time: (i) 18 h, two embryos; (ii) 24 h, two embryos;(iii) 48 h, five embryos. To determine if the medium alone affects the structureof the membrane, control pieces of vitelline membrane were placed on the agar.After the desired length of culture the embryos had attached to the vitellinemembrane by means of cells around the periphery and had expanded radially.The embryos plus vitelline membranes were flushed with chick Ringer solutionto loosen them from the agar and placed into fixative.

RESULTS

The vitelline membrane from freshly laid eggs

The ultrastructure of the chick vitelline membrane taken from various regionsof the egg has been described in detail from thin sections (Bellairs et al. 1963).The membranes of fresh (unincubated) eggs (both fertilized and unfertilized)are composed of two layers (Fig. 2A). The layer next to the albumen (the outerlayer) varies in thickness from 3 to 8-5 fi. It is composed of a variable number offibrous layers lying one above the other in transverse section. The layer next tothe yolk and the embryo (the inner layer) is 1-2 /.t thick in transverse section.Transverse and horizontal (tangential) sections indicate that it consists of ameshwork of fibers 0-1-1 ft in diameter (see Bellairs et al. 1963). The fibers fromfresh eggs usually stain homogeneously with uranyl acetate, lead hydroxide orphosphotungstic acid, although they have been reported occasionally to containfibrils 150-300 A in diameter (Bellairs et al. 1963). A granular 'continuousmembrane' 500-1000 A thick lies between the inner and outer layers. The abovedescription applies to membranes fixed and embedded in the different waysdescribed under Methods.

Carbon replicas of the chick vitelline membrane show that the meshlikestructure seen in tangential sections of the inner layer is also present at its innersurface (Fig. 2B). Several levels of the three-dimensional meshwork are visiblein replicas. They indicate the wide range in fiber diameter and show the great

Avian vitelline membrane 411

Fig. 2. A. Transverse section through the vitelline membrane of an unincubatedchick egg. cm, Continuous membrane;/, cylindrical fibers; //, inner layer; ol, partof outer layer, x .19 600. B. Carbon replica of the inner surface of the vitellinemembrane from an unincubated chick egg. The dark bodies (arrows) probablyrepresent accumulations of carbon particles. /, fibers of the inner layer, x 21 000.

472 C.JENSEN

extent to which fibers branch and coalesce. The surface of the fibers appears tobe smooth. The replicas are of additional interest in indicating the nature of thesurface upon which the early embryo is dependent for normal expansion.

Vitelline membranes from unincubated turkey and pigeon eggs are similar instructure to the chick vitelline membrane, the only difference being the thicknessof the inner layer (turkey, 2-2-5/t; pigeon, 1-1-6 fi) and outer layer (turkey,4-2 fi; pigeon, 10-0/*).

Changes in structure of the inner layer of the chick vitellinemembrane during embryonic development

Unincubated blastoderm, stage 1. The vitelline membrane located over theembryo is identical in structure to membranes from non-embryonic regions ofunincubated eggs, both fertilized and unfertilized (see Fig. 2 A).

9 hours, stage 3 (Fig. 3A). A cross-section through the inner layer locatedover the embryo shows that the structure differs from that in the unincubatedegg. The fibers do not stain homogeneously; instead, lighter staining areas arescattered throughout (compare with Fig. 2 A).

82 hours, stage 21 (Fig. 3B). This represents a period just before the membranenormally ruptures over the embryo. The inner layer of the vitelline membraneover the embryo consists of groups of densely staining units lying within a less-dense matrix. These units are in the form of fibrils, 150-300 A wide with lengthsup to 0-7 ji\ and dots, 250-350 A in diameter. When the block is sectioned in aplane 90° to the original, both dots and fibrils are again visible. It is thereforeprobable that the dots represent cross-sections of fibrils.

99 hours, stage 23 (Fig. 4A). The membrane has ruptured over the embryo,and slipped down to the equatorial region of the egg. The slipped part of themembrane is thrown into tight folds visible with the naked eye. In electronmicrographs of pieces of membrane located next to the point of rupture (origin-ally over the pellucid area of the embryo), the inner layer is thrown into folds,and fibrils and dots are again visible. The outer layer does not exhibit such afolding. (When eggs of slightly younger stages are opened, the vitelline mem-brane has not ruptured. However, it invariably ruptures during manipulationof the yolk and immediately slips down to the equatorial region of the yolk.)

107 hours, stage 24 (Fig. 4B, C) and 134 hours, stage 27. The rupturedvitelline membrane has slipped even farther toward the vegetal pole. The innerlayer near the ruptured area consists of scattered bundles of 150-300 A fibrils.

Prior to 107 h of incubation (samples were taken at 9, 45, 82, 99, and 103 h)the vitelline membrane from the vegetal pole resembles membranes from unincu-bated eggs. At 107 h, however, the inner layer contains less-dense areas similarto the inner layer over a 9 h embryo (see Fig. 3 A). Therefore, though the yolksac reaches the vegetal pole by 96 h of incubation, changes in structure in thevitelline membrane in this area are not visible until approximately 10 h later.

In some specimens (7 out of the 33 examined) incubated for periods ranging

Avian vitelline membrane 473

Fig. 3. A. Transverse section through the inner layer of the vitelline membranelocated directly over a nine-hour chick embryo. Many lighter-staining areas (arrows)are present within the fibers of the inner layer (//). x 19 000. B. Transverse sectionthrough the inner layer of the vitelline membrane located directly over the pellucidarea of an 82 h chick embryo. /, Faint traces of the original fibrous structure; d, dots;fl, fibrils; //, inner layer, x 18 200.

474 C.JENSEN

Avian vitelline membrane Al5

from 9 to 107 h, fibrillar bundles appear closer together in regions over theembryo and appear to form an almost continuous fibrillar layer (Fig. 5 A). Thetotal width (1-3 /<) of this layer, designated as continuous, is the same as thefibrous inner layer of the unincubated egg. In horizontal sections the continuousinner layer appears to consist of bundles of fibrils (Fig. 5B). Fine threads 75 Ain diameter are present between the fibrillar bundles as well as between indivi-dual fibrils, with which they occasionally appear to be continuous. Regions ofsome specimens show what may be transitional stages between a fibrous typeof layer and a continuous layer; the outlines of structures resembling inner layerfibers are visible in cross-sections (Fig. 5A).

Replicas of the inner surface of the inner layer from incubated chick vitellinemembranes also reveal ultrastructural changes. At 84 h of incubation (Fig. 6 A)the fibers of the inner layer located over the embryo appear to be pitted withholes 400-1000 A in diameter; and in some areas fibrils 250-400 A in diameterare visible (compare with the inner surface of an unincubated chick vitellinemembrane in Fig. 2B). It was not possible to obtain a satisfactory replica of theinner layer surface of later developmental stages, perhaps because of foldingof the inner layer after the membrane ruptures.

The continuous membrane between inner and outer layers is also sometimesaltered during development. In some instances it is present (Fig. 4A), while inothers it is not visible (Figs. 3 A, B, 4B).

The inner layer from different regions of the same egg

The same sequence of changes occurs in pieces of vitelline membrane fromdifferent regions of an incubated egg. In an egg incubated for 45 h (stage 11)the expanding yolk sac cells have not yet reached the vegetal pole (refer toFig. 1). The inner layer from the vegetal region is similar in structure to theunincubated egg vitelline membrane (see Fig. 2A); it is composed of homo-geneously stained fibers. Lighter-staining areas are present in the inner layerfibers located over the opaque area of the embryo (as in Fig. 3A), and the fibersin the inner layer located over the pellucid area (which has been in contact withembryonic cells for a longer period) are composed of fibrils (as in Fig. 3B).

Fig. 4. A. Transverse section through the inner layer of the vitelline membrane froma chick egg incubated for 99 h. The membrane has broken over the embryo andslipped toward the vegetal pole. The region pictured here was located over thepellucid area of the embryo before the membrane ruptured. The inner layer (//) isthrown into folds, and fibrils are visible within it. cm, continuous membrane; ol, partof outer layer, x 7420. B. Transverse section through the vitelline membrane froma chick egg incubated for 107 h. The membrane has ruptured. The region picturedhere was originally located over the pellucid area of the embryo. The remnants offibers (/) are sometimes visible. Fibrils (fl) are very evident within the inner layer(//); olf, fibrils of the outer layer, x 13 300. C. Enlargement of part of the samemembrane shown in B. Arrows indicate fibrils in the inner layer, x 56 700.

476 C.JENSEN

• — " 'M.hs^ • •

Avian vitelline membrane All

Changes in the outer layer of the chick vitelline membrane

Changes in structure of the outer layer are not as consistent as those in theinner layer. In some membranes from incubated eggs the outer layer threadsappear thicker (Fig. 5A) or thinner (Fig. 4B) than they are in the unincubatedegg. However, in other incubated specimens, the outer layer is identical instructure to this layer in the unincubated egg (Fig. 4A).

In spite of marked structural changes in the inner layer during development,the thickness of both the inner and outer layers does not change significantly,and remains within the range of dimensions found in vitelline membranes ofunincubated eggs.

Changes in structure of pigeon and turkey vitellinemembranes during embryonic development

Similar structural changes also occur in the vitelline membranes of incubatedpigeon and turkey eggs. The fibers in the inner layer of membranes located over10, 48, and 72 h pigeon embryos (Fig. 6B) consist of fibrils.

Changes in the turkey vitelline membrane parallel the slower development ofturkey embryos (28 days to hatching instead of 21 in the chick). The vitellinemembrane ruptures over the turkey embryo between the fifth and sixth days ofdevelopment, 2 days later than in the chick. As early as 48 h of development(stage 9) the inner layer located over the embryo is modified in structure. Twoof the 14 membranes examined have fibrillar inner layers similar to the chick(see Fig. 3B). In the majority of membranes the inner layer over the embryo iscontinuous, with fibrils and dots scattered throughout a less-dense matrix(Fig. 6C). The width of the fibrils ranges from 200 to 400 A, with lengthsobserved up to 0-3 JLC. The inner layer from the vegetal pole of the latest stagesexamined, 186 h (stage 27), is no different in structure from the unincubated egg;homogeneously staining fibers are present.

In vitro changesIdentical changes in ultrastructure are produced by culturing a chick embryo

on a piece of chick vitelline membrane in vitro. The inner layer located next tothe center of the embryo after 18, 24 and 48 h of culture resembles that alreadydescribed after incubation in ovo. In some cases fibrils 140-170 A in width aregrouped into fibers (as in Fig. 3B); in other cases they form the continuous typeof inner layer (as in Fig. 5A).

Fig. 5. A. Transverse section through the vitelline membrane located over a 62 hchick embryo. The inner layer (//) is of the continuous type. In one area which mayrepresent a transitional stage from the fibrous type (shown in Fig. 2 A) to the con-tinuous type, several fibers (/) are visible; ol, part of outer layer, x 26 500. B.Horizontal (tangential) section through the inner layer of the vitelline membranepictured in A. Arrows indicate fibrils, which sometimes form fibrillar bundles(brackets). A threadlike material (/) fills the gaps between bundles, x 43 700.

478 C. JENSEN

Avian vitelline membrane Al9

Inner layer fibers in pieces next to the edge of the embryo contain less-denseareas (as in Fig. 3A), while areas not covered by the embryo and control piecesresemble the vitelline membrane from an unincubated egg.

DISCUSSION

The present electron microscope studies suggest that there are progressiveultrastructural changes in the inner layer of the vitelline membrane duringdevelopment. The changes involve, first, the appearance of lighter staining areasin the originally dense, homogeneously staining fibers of the inner layer (Fig.3A), followed by the appearance of fibrils within the inner layer fibers (Figs.3B, 4A-C). These changes in the membrane could be preparation artifacts.The variations in structure seen in the inner layer during development (suchas the continuous type of inner layer) suggest this possibility. However, the useof different fixatives (glutaraldehyde and osmium tetroxide), buffers (Veronaland phosphate), embedding media (Vestopal and Araldite) and stains (uranylacetate, lead hydroxide, phosphotungstic acid and no stain) reveal that noparticular structural change can be correlated with a certain preparative pro-cedure. Also, both the continuous type of inner layer and the non-continuoustype appeared in different specimens which had been incubated the same lengthof time and were fixed and embedded at the same time. In addition, surfacereplicas of the membrane, which are prepared differently from thin sections,reveal structural changes during development.

Perhaps the most convincing evidence that the observed changes are relatedto actual structural ones is the observation that structural changes are alwaysvisible in pieces which have been in contact with embryonic cells for at least 9 h,while pieces from the same egg which have not been in contact with the embryoare not altered, i.e. they resemble the inner layer from unincubated eggs.

Therefore, the fibrils seen in the inner layer over the embryo most likelyrepresent actual structures rather than artifacts. These fibrils may be present atall times in the inner layer, as they are occasionally visible in the inner layersof some unincubated avian eggs. They have been observed in unincubatedparakeet vitelline membranes stained with uranyl acetate and in one unincu-bated turkey vitelline membrane (Jensen, 1966). Also, Bellairs et al. (1963) notedin the inner layer of some chick vitelline membranes discrete fibrils similar in

Fig. 6. A. Carbon replica of the inner surface of the vitelline membrane located overthe pellucid area of an 84 h chick embryo. The fibers (/) of the inner layer appear tobe pitted with holes (arrows). Occasional fibrils (fl) are visible, x 21 400. B. Trans-verse section through part of the inner layer of the vitelline membrane located overthe pellucid area of a pigeon embryo. The fibers (/) are composed of fibrils, x 43 500.C. Transverse section through the vitelline membrane located over a 48 h turkeyembryo. The inner layer (//) is continuous and fibrils (arrows) are visible; ol, outerlayer, x 21 600.31 J E E M 2 1

480 C. JENSEN

appearance to the fibrils seen in connexion with the developing embryo in thepresent studies.

If one assumes that fibrils are present but usually not distinguishable in theinner layer of the unincubated vitelline membrane, their subsequent appearanceduring development could result from one of two possible mechanisms. First,the fibrils may be masked in the unincubated vitelline membrane because oftheir inability to take up more stain than the surrounding matrix. A change inproperties of the matrix around the fibrils may occur during development sothat the matrix is either dissolved or stains less than the fibrils, resulting in theirbecoming visible. This possibility is suggested by the appearance of lighterstaining areas within the inner layer fibers, which is the first visible change inthe inner layer during development (Fig. 3 A). A second possibility is that thefibrous meshwork of the inner layer of the unincubated egg is composed ofbundles of fibrils so tightly packed that individual fibrils are not visible; instead,the entire bundle would stain as a homogeneous structure, which has beendesignated as a fiber. During development the fibrils in the inner layer maybecome less tightly packed, and consequently visible within the fibers (Figs. 3B,4A-C). The presence of pits in the fibers in surface replicas of incubatedvitelline membranes (Fig. 6A) suggests that there are actually spaces within thefibers (between fibrils), indicating a dispersal of fibrillar material or the dis-appearance of a matrix surrounding fibrils rather than a differential staining ofthe matrix.

The presence of a continuous type of inner layer (Fig. 5 A) strongly suggeststhat the fibrils have dispersed and filled in most of the gaps which were originallypresent between fibers in the unincubated egg. That these are actually gaps andnot merely material which has taken up no stain is shown in the replica (Fig. 2B)as well as by the fact that the embryonic cells send processes into them (Bellairs,1963). The continuous type could not result from a swelling of the entire innerlayer since the total thickness of the continuous inner layer is the same as thatof the inner layer from the unincubated egg.

Since the inner layer of the vitelline membrane is predominantly protein(Bellairs et al. 1963), the changes in arrangement of the fibrils within it mayresemble the aggregation and dispersion of other fibrillar proteins such ascollagen (Gross, Lapiere & Tanzer, 1963). The first indication of the inner layerin immature ovarian eggs is the appearance of fibrils 100 A in diameter (Bellairs,1965; Wyburn, Aitken & Johnston, 1965). The characteristic meshwork ofhomogeneous fibers appears in the inner layer only during later stages ofoogenesis and may result from an aggregation of the fibrils into bundles duringoogenesis. The fibrils which appear in the immature ovarian egg may be thesame ones visible within the inner layer during embryonic development. Conse-quently, changes in the vitelline membrane during embryonic development maymirror in reverse the formation of the vitelline membrane in the ovary.

Because the structural changes of the vitelline membrane occur only where the

Avian vitelline membrane 481

membrane has been in contact with the embryo, the embryonic cells may beresponsible for the changes. The close association of the cells with the innersurface of the membrane makes it possible for interactions between embryoniccells and vitelline membrane to occur. The fact that the structure of the innerlayer is altered by culturing an embryo on a piece of vitelline membrane in vitrois strong evidence for the role of the embryo in causing structural changes in themembrane. The in vitro experiments also eliminate the possibility that stretchingof the vitelline membrane during development due to swelling of the yolk(Romanoff & Romanoff, 1949) causes changes in its structure. Additionalevidence that the embryo (rather than the process of incubation) is involved inthe structural changes of the inner layer comes from the fact that the innerlayer remains unchanged in structure in unfertilized eggs incubated at 38 °C forperiods up to 14 days (Jensen, 1966). The inner layer of fertile eggs stored at4 °C for periods as long as 6 months also remains unchanged in structure(Jensen, 1966).

The present studies indicate that approximately 10 h of contact betweenvitelline membrane and chick embryo are required before changes in structureof the inner layer are visible. In the turkey longer and more variable periods ofcontact between embryonic cells and vitelline membrane appear to be necessaryfor changes in structure to occur. The vitelline membrane at the vegetal pole isnot modified after 186 h of incubation, even though the embryonic cells reachthe vegetal pole by 115 h of incubation. However, the area over the embryo(at the animal pole) is modified in structure after 48 h.

Since the vitelline membrane is under tension and its mechanical strengthdecreases progressively, it eventually ruptures in the weakest region (at theanimal pole). The altered area of the membrane with dispersed fibrils may beweaker than the fresh vitelline membrane, perhaps because of ruptured bondsbetween fibrils. The immediate slipping down of the vitelline membrane overthe yolk after it ruptures and the folding of the inner layer near the point ofrupture (Fig. 4A) indicates its elasticity (according to Moran (1936) the vitellinemembrane actually increases in elasticity during incubation). Swelling of theyolk due to passage into it of water during development (Romanoff &Romanoff, 1949) may increase the tension upon the membrane.

The fact that the outer layer of the vitelline membrane is not visibly alteredin structure during development might suggest that the strength of the innerlayer is solely responsible for maintaining the membrane intact in the egg.However, if the outer layer is destroyed by various treatments which do notappear to affect the inner layer (i.e. incubation of infertile eggs, incubation ofpieces of membrane in albumen), the mechanical strength of the membrane isdrastically reduced (Jensen, 1966). Therefore, it appears that both the inner andouter layers are important in maintaining the mechanical strength of thevitelline membrane and that breakdown of either results in a loss of strength.

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SUMMARY

1. The inner layer of the vitelline (yolk) membrane of the unincubated chickenegg is composed of a meshwork of homogeneously staining fibers. Duringdevelopment there are progressive structural changes in this layer which appearto be brought about by the embryonic cells.

2. The structural changes involve the appearance of lighter staining areaswithin the fibers of the inner layer, followed by the appearance of fibrils150-300 A in diameter within the fibers.

3. In some specimens the fibrils are dispersed throughout the entire innerlayer, resulting in a continuous fibrillar inner layer. Some specimens show areaswhich appear to be intermediates between a non-continuous and a continuoustype of inner layer.

4. Structural changes occur only in regions of the membrane which havebeen in contact with the embryo for at least 10 h.

5. Identical changes are produced by culturing chick embryos on pieces ofvitelline membrane in vitro. Similar structural changes also occur in theinner layers of vitelline membranes from incubated fertilized pigeon andturkey eggs.

6. It is suggested that the embryonic cells are responsible for structuralalterations in the vitelline membrane which weaken the membrane and subse-quently cause it to rupture over the embryo.

RESUME

Modifications ultrastructurales de la membrane vitelline desOiseaux pendant le developpement embryonnaire

1. La couche interne de la membrane vitelline (cote jaune) de l'ceuf nonincube du Poulet est formee par un reseau de fibres se colorant homogenement.Pendant le developpement embryonnaire, des modifications progressives de lastructure interviennent dans cette couche. Elles semblent liees a la presence descellules embryonnaires.

2. Les transformations structurales se manifestent, dans les fibres de lacouche interne, par l'apparition de zones plus faiblement colorees et secondaire-ment par l'apparition de fibrilles de 150 a 300 A de diametre.

3. Dans quelques cas, les fibrilles sont dispersees dans toute la couche interne,formant ainsi une couche fibrillaire interne continue. Quelques specimenspresentent des zones, dont l'aspect est intermediaire entre celui d'une coucheinterne 'continue' et celui d'une couche interne 'non continue'.

4. Les changements structuraux surviennent seulement dans les regionsplacees au contact de l'embryon pendant au moins 10 heures.

5. Des changements analogues interviennent en cultivant in vitro desembryons de Poulet sur des fragments de membrane vitelline. Des transforma-

Avian vitelline membrane 483

tions identiques se produisent egalement dans la couche interne de la membranevitelline de Foeuf feconde et incube de Pigeon et de Dindon.

6. Ces donnees suggerent que les cellules embryonnaires sont responsablesdes alterations structurales de la membrane vitelline. Ces alterations, en dimi-nuant la resistance de la membrane, entraineraient sa rupture au-dessus derembryon.

This study represents a portion of a Doctoral thesis presented to the Graduate School ofthe University of Minnesota while the author was supported by a Predoctoral Fellowshipfrom the National Institutes of Health, U.S. Public Health Service. This investigation wasalso supported in part by an Institutional Grant from the American Cancer Society.

The author is especially grateful to Dr Ruth Bellairs for providing many helpful suggestionstoward preparation of this manuscript. Appreciation is also extended to Dr Nelson T. Spratt,Jr., for advice during the course of the research and to Dr Philip Grant for reviewing themanuscript.

REFERENCES

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BELLAIRS, R. (1963). Differentiation of the yolk sac of the chick studied by electron micro-scopy. /. Embryol. exp. Morph. 7, 146-64.

BELLAIRS, R. (1965). The relationship between oocyte and follicle in the hen's ovary as shownby electron microscopy. / . Embryol. exp. Morph. 13, 215-33.

BELLAIRS, R., HARKNESS, M. & HARKNESS, R. D. (1963). The vitelline membrane of the hen'segg: a chemical and electron microscopical study. / . Ultrastruct. Res. 8, 339-59.

BRADLEY, D. E. (1960). Replica techniques in applied electron microscopy. / / R. microsc.Soc. 79, 101-18.

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(Manuscript received 14 October 1968)