Ultrastructural Changes in the Avian Vitelline Membrane During Embryonic Development

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

Ultrastructural changes in the avian vitelline membrane 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 early embryonic development; it encloses the yolk and provides a substratum for expansion of the embryo (Fig. 1). Expansion appears to be dependent upon the movement of cells at the edge of the blastoderm which is intimately associated with the inner layer of the vitelline membrane (New, 1959; Bellairs, 1963). The blastoderm (embryonic plus extraembryonic cells) has almost covered the entire surface of the yolk by the third and fourth days of incubation, and when this stage has been reached the vitelline membrane ruptures over the embryo and slips toward the vegetal pole.

Embryonic axis Pellucid area

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

Yolk

Vitelline membrane

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 consequence of a decrease in its mechanical strength (Moran, 1936), which changes most rapidly 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. 468 C.JENSEN mechanical strength of the membrane is decreased by 73 % at the animal pole and only 37 % at the vegetal pole (Mesarosh, 1957). The factors responsible for weakening of the membrane during development have not been determined. Romanoff & Romanoff (1949) suggested that diffu- sion of water from the albumen into the yolk during incubation of eggs causes the yolk to swell, stretching and weakening the vitelline membrane. Loss of strength 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 cells change the nature of adjacent areas of the vitelline membrane with which they are in close association. Since weakening of the membrane is greatest in the area located over the embryo, several investigators have suggested that the cells may secrete an enzyme similar to the hatching proteases of invertebrates and lower vertebrates which digests the vitelline membrane (Moran, 1936; Ragozina, 1957). Bellairs, Harkness & Harkness (1963) suggested that during development structural changes in the membrane accompany the decrease in mechanical strength. As they found no changes in thickness of the vitelline membrane during incubation, they suggested that the decrease in mechanical strength is a result of qualitative changes in the membrane rather than quantitative ones. Although histochemical changes are known to occur in the vitelline membrane during early development (Shalumovich, 1960), no structural changes have been reported. The present study indicates that there are progressive ultrastructural changes in the vitelline membrane during development which correlate with changes in the strength of the membrane and may be produced through activities of the embryonic cells.

MATERIALS Pieces of vitelline membrane which had been in contact with embryonic cells for varying lengths of time were selected for observation (refer to Fig. 1). Since the blastoderm expands radially during development, a piece of vitelline membrane located directly over the embryonic axis or pellucid area has been in contact 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 membrane from the vegetal pole has not yet been in contact with embryonic cells. The yolk sac reaches the vegetal pole at approximately 96 h of incubation; therefore a piece of vitelline membrane taken from the vegetal pole after 106 h of incubation has been in contact with embryonic cells for 10 h. Consequently, there are two ways to measure the amount of time the cells have been in contact with the vitelline membrane: (1) one may take pieces of vitelline membrane from analogous areas in eggs of different stages of incubation, or (2) one may take pieces of membrane from different parts of the same incubated egg. Vitelline membranes 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 two unfertilized. (ii) Thirty-three fertilized chicken eggs incubated at 38 °C for periods ranging from 9 to 134 h. (iii) Six freshly laid turkey (Broad-White-Nicholas) eggs; four were fertilized and two unfertilized. (iv) Fourteen fertilized turkey eggs incubated at 38 °C for periods ranging from 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 microscopy The 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 membrane (usually from the animal pole, vegetal pole and over the peripheral edge of the blastoderm) were cut out with scissors and transferred to a Petri dish containing fresh Ringer solution. Adhering yolk and albumen were quickly removed with forceps and the pieces of membrane were placed in the fixative. Several membranes were rinsed in sodium Veronal or 01 M phosphate buffers rather than Ringer solution. The majority of specimens were fixed for 1 h in 1 % ice-cold osmium tetroxide buffered with sodium Veronal to a pH of 7-4 (Palade, 1952). Some specimens were fixed for 30min in 4 % glutaraldehyde buffered 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 buffered to pH 7-4 with phosphate buffer. After fixation in the cold, most specimens were rapidly dehydrated in an acetone series, and embedded in Vestopal W (Martin Jaeger, Geneva, Switzerland). Other material was dehydrated in an alcohol series, and embedded in Araldite (Glauert & Glauert, 1958). Some specimens were stained for 20 min to 1 h in 1 % phosphotungstic acid in absolute alcohol after dehydration. Thin sections were cut on a Porter-Blum microtome with glass knives and picked up on formvar-covered grids which had been stabilized by a light coating of evaporated carbon. Most sections were stained by floating the grids on the surface of a 2 % aqueous solution of uranyl acetate for 10-30 min at room temperature. Others were stained with uranyl acetate followed by lead hydroxide for 20 min. All pictures were taken with an RCA EMU-3D electron microscope operated at 50 KV. AH electron micrographs in this paper are of material fixed in osmium tetroxide, embedded in Vestopal W, and stained with uranyl acetate. Material prepared in the other ways mentioned above did not appear to differ from the material 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 most consistent results. The two-stage method used involved making a primary formvar impression of the fixed vitelline membrane and a secondary carbon replica which was placed on a grid and examined in the electron microscope.

C. Culture of chick embryos on pieces of vitelline membrane The methods used for culture of embryos on the surface of the vitelline membrane are identical to those described by Spratt (1963). Ten-hour chick embryos were explanted with their ectodermal surface against the inner surface of vitelline membranes on a solid agar-chick extract medium and incubated for the 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 structure of the membrane, control pieces of vitelline membrane were placed on the agar. After the desired length of culture the embryos had attached to the vitelline membrane by means of cells around the periphery and had expanded radially. The embryos plus vitelline membranes were flushed with chick Ringer solution to 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 regions of 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 outer layer) varies in thickness from 3 to 8-5 fi. It is composed of a variable number of fibrous layers lying one above the other in transverse section. The layer next to the 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 a meshwork of fibers 0-1-1 ft in diameter (see Bellairs et al. 1963). The fibers from fresh eggs usually stain homogeneously with uranyl acetate, lead hydroxide or phosphotungstic acid, although they have been reported occasionally to contain fibrils 150-300 A in diameter (Bellairs et al. 1963). A granular 'continuous membrane' 500-1000 A thick lies between the inner and outer layers. The above description applies to membranes fixed and embedded in the different ways described under Methods. Carbon replicas of the chick vitelline membrane show that the meshlike structure seen in tangential sections of the inner layer is also present at its inner surface (Fig. 2B). Several levels of the three-dimensional meshwork are visible in 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 unincubated chick egg. cm, Continuous membrane;/, cylindrical fibers; //, inner layer; ol, part of outer layer, x .19 600. B. Carbon replica of the inner surface of the vitelline membrane from an unincubated chick egg. The dark bodies (arrows) probably represent 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 to be smooth. The replicas are of additional interest in indicating the nature of the surface upon which the early embryo is dependent for normal expansion. Vitelline membranes from unincubated turkey and pigeon eggs are similar in structure to the chick vitelline membrane, the only difference being the thickness of 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 vitelline membrane during embryonic development Unincubated blastoderm, stage 1. The vitelline membrane located over the embryo is identical in structure to membranes from non-embryonic regions of unincubated eggs, both fertilized and unfertilized (see Fig. 2 A). 9 hours, stage 3 (Fig. 3A). A cross-section through the inner layer located over the embryo shows that the structure differs from that in the unincubated egg. The fibers do not stain homogeneously; instead, lighter staining areas are scattered throughout (compare with Fig. 2 A). 82 hours, stage 21 (Fig. 3B). This represents a period just before the membrane normally ruptures over the embryo. The inner layer of the vitelline membrane over 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 lengths up to 0-7 ji\ and dots, 250-350 A in diameter. When the block is sectioned in a plane 90° to the original, both dots and fibrils are again visible. It is therefore probable 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 the membrane is thrown into tight folds visible with the naked eye. In electron micrographs of pieces of membrane located next to the point of rupture (originally 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 a folding. (When eggs of slightly younger stages are opened, the vitelline membrane has not ruptured. However, it invariably ruptures during manipulation of 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 ruptured vitelline membrane has slipped even farther toward the vegetal pole. The inner layer 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 unincubated eggs. At 107 h, however, the inner layer contains less-dense areas similar to the inner layer over a 9 h embryo (see Fig. 3 A). Therefore, though the yolk sac reaches the vegetal pole by 96 h of incubation, changes in structure in the vitelline 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 membrane located 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 section through the inner layer of the vitelline membrane located directly over the pellucid area 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 the embryo and appear to form an almost continuous fibrillar layer (Fig. 5 A). The total width (1-3 /<) of this layer, designated as continuous, is the same as the fibrous inner layer of the unincubated egg. In horizontal sections the continuous inner layer appears to consist of bundles of fibrils (Fig. 5B). Fine threads 75 A in diameter are present between the fibrillar bundles as well as between individual fibrils, with which they occasionally appear to be continuous. Regions of some specimens show what may be transitional stages between a fibrous type of layer and a continuous layer; the outlines of structures resembling inner layer fibers are visible in cross-sections (Fig. 5A). Replicas of the inner surface of the inner layer from incubated chick vitelline membranes 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 with holes 400-1000 A in diameter; and in some areas fibrils 250-400 A in diameter are visible (compare with the inner surface of an unincubated chick vitelline membrane in Fig. 2B). It was not possible to obtain a satisfactory replica of the inner layer surface of later developmental stages, perhaps because of folding of the inner layer after the membrane ruptures. The continuous membrane between inner and outer layers is also sometimes altered during development. In some instances it is present (Fig. 4A), while in others 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 from different 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 to Fig. 1). The inner layer from the vegetal region is similar in structure to the unincubated egg vitelline membrane (see Fig. 2A); it is composed of homogeneously stained fibers. Lighter-staining areas are present in the inner layer fibers located over the opaque area of the embryo (as in Fig. 3A), and the fibers in the inner layer located over the pellucid area (which has been in contact with embryonic 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 from a chick egg incubated for 99 h. The membrane has broken over the embryo and slipped toward the vegetal pole. The region pictured here was located over the pellucid area of the embryo before the membrane ruptured. The inner layer (//) is thrown into folds, and fibrils are visible within it. cm, continuous membrane; ol, part of outer layer, x 7420. B. Transverse section through the vitelline membrane from a chick egg incubated for 107 h. The membrane has ruptured. The region pictured here was originally located over the pellucid area of the embryo. The remnants of fibers (/) 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 same membrane shown in B. Arrows indicate fibrils in the inner layer, x 56 700. 476 C.JENSEN

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^ •• 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 the inner layer. In some membranes from incubated eggs the outer layer threads appear thicker (Fig. 5A) or thinner (Fig. 4B) than they are in the unincubated egg. However, in other incubated specimens, the outer layer is identical in structure 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 of unincubated eggs. Changes in structure of pigeon and turkey vitelline membranes during embryonic development Similar structural changes also occur in the vitelline membranes of incubated pigeon and turkey eggs. The fibers in the inner layer of membranes located over 10, 48, and 72 h pigeon embryos (Fig. 6B) consist of fibrils. Changes in the turkey vitelline membrane parallel the slower development of turkey embryos (28 days to hatching instead of 21 in the chick). The vitelline membrane ruptures over the turkey embryo between the fifth and sixth days of development, 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. Two of 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 is continuous, with fibrils and dots scattered throughout a less-dense matrix (Fig. 6C). The width of the fibrils ranges from 200 to 400 A, with lengths observed up to 0-3 JLC. The inner layer from the vegetal pole of the latest stages examined, 186 h (stage 27), is no different in structure from the unincubated egg; homogeneously staining fibers are present.

In vitro changes Identical changes in ultrastructure are produced by culturing a chick embryo on a piece of chick vitelline membrane in vitro. The inner layer located next to the center of the embryo after 18, 24 and 48 h of culture resembles that already described after incubation in ovo. In some cases fibrils 140-170 A in width are grouped into fibers (as in Fig. 3B); in other cases they form the continuous type of inner layer (as in Fig. 5A).

Fig. 5. A. Transverse section through the vitelline membrane located over a 62 h chick embryo. The inner layer (//) is of the continuous type. In one area which may represent a transitional stage from the fibrous type (shown in Fig. 2 A) to the continuous type, several fibers (/) are visible; ol, part of outer layer, x 26 500. B. Horizontal (tangential) section through the inner layer of the vitelline membrane pictured 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-dense areas (as in Fig. 3A), while areas not covered by the embryo and control pieces resemble the vitelline membrane from an unincubated egg.

DISCUSSION The present electron microscope studies suggest that there are progressive ultrastructural changes in the inner layer of the vitelline membrane during development. The changes involve, first, the appearance of lighter staining areas in 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 (such as the continuous type of inner layer) suggest this possibility. However, the use of different fixatives (glutaraldehyde and osmium tetroxide), buffers (Veronal and phosphate), embedding media (Vestopal and Araldite) and stains (uranyl acetate, lead hydroxide, phosphotungstic acid and no stain) reveal that no particular structural change can be correlated with a certain preparative pro- cedure. Also, both the continuous type of inner layer and the non-continuous type appeared in different specimens which had been incubated the same length of time and were fixed and embedded at the same time. In addition, surface replicas 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 related to actual structural ones is the observation that structural changes are always visible 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 embryo are not altered, i.e. they resemble the inner layer from unincubated eggs. Therefore, the fibrils seen in the inner layer over the embryo most likely represent actual structures rather than artifacts. These fibrils may be present at all times in the inner layer, as they are occasionally visible in the inner layers of some unincubated avian eggs. They have been observed in unincubated parakeet vitelline membranes stained with uranyl acetate and in one unincubated turkey vitelline membrane (Jensen, 1966). Also, Bellairs et al. (1963) noted in 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 over the pellucid area of an 84 h chick embryo. The fibers (/) of the inner layer appear to be 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 over the 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 turkey embryo. The inner layer (//) is continuous and fibrils (arrows) are visible; ol, outer layer, x 21 600. 31 JEEM 21 480 C. JENSEN appearance to the fibrils seen in connexion with the developing embryo in the present studies. If one assumes that fibrils are present but usually not distinguishable in the inner layer of the unincubated vitelline membrane, their subsequent appearance during development could result from one of two possible mechanisms. First, the fibrils may be masked in the unincubated vitelline membrane because of their inability to take up more stain than the surrounding matrix. A change in properties of the matrix around the fibrils may occur during development so that the matrix is either dissolved or stains less than the fibrils, resulting in their becoming visible. This possibility is suggested by the appearance of lighter staining areas within the inner layer fibers, which is the first visible change in the inner layer during development (Fig. 3 A). A second possibility is that the fibrous meshwork of the inner layer of the unincubated egg is composed of bundles of fibrils so tightly packed that individual fibrils are not visible; instead, the entire bundle would stain as a homogeneous structure, which has been designated as a fiber. During development the fibrils in the inner layer may become less tightly packed, and consequently visible within the fibers (Figs. 3B, 4A-C). The presence of pits in the fibers in surface replicas of incubated vitelline membranes (Fig. 6A) suggests that there are actually spaces within the fibers (between fibrils), indicating a dispersal of fibrillar material or the dis- appearance of a matrix surrounding fibrils rather than a differential staining of the matrix. The presence of a continuous type of inner layer (Fig. 5 A) strongly suggests that the fibrils have dispersed and filled in most of the gaps which were originally present between fibers in the unincubated egg. That these are actually gaps and not 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 inner layer since the total thickness of the continuous inner layer is the same as that of 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 may resemble the aggregation and dispersion of other fibrillar proteins such as collagen (Gross, Lapiere & Tanzer, 1963). The first indication of the inner layer in immature ovarian eggs is the appearance of fibrils 100 A in diameter (Bellairs, 1965; Wyburn, Aitken & Johnston, 1965). The characteristic meshwork of homogeneous fibers appears in the inner layer only during later stages of oogenesis and may result from an aggregation of the fibrils into bundles during oogenesis. The fibrils which appear in the immature ovarian egg may be the same ones visible within the inner layer during embryonic development. Conse- quently, changes in the vitelline membrane during embryonic development may mirror 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 be responsible for the changes. The close association of the cells with the inner surface of the membrane makes it possible for interactions between embryonic cells and vitelline membrane to occur. The fact that the structure of the inner layer is altered by culturing an embryo on a piece of vitelline membrane in vitro is strong evidence for the role of the embryo in causing structural changes in the membrane. The in vitro experiments also eliminate the possibility that stretching of the vitelline membrane during development due to swelling of the yolk (Romanoff & Romanoff, 1949) causes changes in its structure. Additional evidence that the embryo (rather than the process of incubation) is involved in the structural changes of the inner layer comes from the fact that the inner layer remains unchanged in structure in unfertilized eggs incubated at 38 °C for periods up to 14 days (Jensen, 1966). The inner layer of fertile eggs stored at 4 °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 between vitelline membrane and chick embryo are required before changes in structure of the inner layer are visible. In the turkey longer and more variable periods of contact between embryonic cells and vitelline membrane appear to be necessary for changes in structure to occur. The vitelline membrane at the vegetal pole is not modified after 186 h of incubation, even though the embryonic cells reach the 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 strength decreases progressively, it eventually ruptures in the weakest region (at the animal pole). The altered area of the membrane with dispersed fibrils may be weaker than the fresh vitelline membrane, perhaps because of ruptured bonds between fibrils. The immediate slipping down of the vitelline membrane over the yolk after it ruptures and the folding of the inner layer near the point of rupture (Fig. 4A) indicates its elasticity (according to Moran (1936) the vitelline membrane actually increases in elasticity during incubation). Swelling of the yolk 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 altered in structure during development might suggest that the strength of the inner layer is solely responsible for maintaining the membrane intact in the egg. However, if the outer layer is destroyed by various treatments which do not appear to affect the inner layer (i.e. incubation of infertile eggs, incubation of pieces of membrane in albumen), the mechanical strength of the membrane is drastically reduced (Jensen, 1966). Therefore, it appears that both the inner and outer layers are important in maintaining the mechanical strength of the vitelline membrane and that breakdown of either results in a loss of strength.

31-2 482 C.JENSEN

SUMMARY 1. The inner layer of the vitelline (yolk) membrane of the unincubated chicken egg is composed of a meshwork of homogeneously staining fibers. During development there are progressive structural changes in this layer which appear to be brought about by the embryonic cells. 2. The structural changes involve the appearance of lighter staining areas within the fibers of the inner layer, followed by the appearance of fibrils 150-300 A in diameter within the fibers. 3. In some specimens the fibrils are dispersed throughout the entire inner layer, resulting in a continuous fibrillar inner layer. Some specimens show areas which appear to be intermediates between a non-continuous and a continuous type of inner layer. 4. Structural changes occur only in regions of the membrane which have been in contact with the embryo for at least 10 h. 5. Identical changes are produced by culturing chick embryos on pieces of vitelline membrane in vitro. Similar structural changes also occur in the inner layers of vitelline membranes from incubated fertilized pigeon and turkey eggs. 6. It is suggested that the embryonic cells are responsible for structural alterations 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 des Oiseaux pendant le developpement embryonnaire 1. La couche interne de la membrane vitelline (cote jaune) de l'ceuf non incube du Poulet est formee par un reseau de fibres se colorant homogenement. Pendant le developpement embryonnaire, des modifications progressives de la structure interviennent dans cette couche. Elles semblent liees a la presence des cellules embryonnaires. 2. Les transformations structurales se manifestent, dans les fibres de la couche 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 specimens presentent des zones, dont l'aspect est intermediaire entre celui d'une couche interne 'continue' et celui d'une couche interne 'non continue'. 4. Les changements structuraux surviennent seulement dans les regions placees au contact de l'embryon pendant au moins 10 heures. 5. Des changements analogues interviennent en cultivant in vitro des embryons 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 membrane vitelline de Foeuf feconde et incube de Pigeon et de Dindon. 6. Ces donnees suggerent que les cellules embryonnaires sont responsables des alterations structurales de la membrane vitelline. Ces alterations, en dimi- nuant la resistance de la membrane, entraineraient sa rupture au-dessus de rembryon. This study represents a portion of a Doctoral thesis presented to the Graduate School of the University of Minnesota while the author was supported by a Predoctoral Fellowship from the National Institutes of Health, U.S. Public Health Service. This investigation was also 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 suggestions toward 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 the manuscript. REFERENCES BALLS, A. K. & SWENSON, T. L. (1934). Proteolysis in stored eggs. Ind. Engng Chem. 26, 570-2. BELLAIRS, R. (1963). Differentiation of the yolk sac of the chick studied by electron microscopy. /. Embryol. exp. Morph. 7, 146-64. BELLAIRS, R. (1965). The relationship between oocyte and follicle in the hen's ovary as shown by electron microscopy. /. Embryol. exp. Morph. 13, 215-33. BELLAIRS, R., HARKNESS, M. & HARKNESS, R. D. (1963). The vitelline membrane of the hen's egg: a chemical and electron microscopical study. /. Ultrastruct. Res. 8, 339-59. BRADLEY, D. E. (1960). 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(Manuscript received 14 October 1968)