OBSERVATIONS ON THE DEVELOPMENT AND STRUOTURE OF THE VITELLINE MEMBRANE OF THE HEN'S EGG: AN ELEOTRON MIOROSOOPE STUDY

By JOAN M. BAIN* and JANICE M. HALL*

[Manuscript received December 9, 1968]

Summary Stages in the development of the outer layer of the vitelline membrane of a hen's egg have been observed in an egg found in the infundibulum of a sacrificed White Leghorn hen. Tissue from the infundibulum and the underlying egg yolk material was taken at increasing distances from the upper end of the egg and the relationship between the secretory cells of the infundibulum and the vitelline mem­ brane observed. The structure of the vitelline membrane in ova just liberated from the ovary and not yet in the oviduct and that of the vitelline membrane in new-laid eggs from other White Leghorn hens were observed for comparison.

1. INTRODUOTION Bellairs, Harkness, and Harkness (1963) investigated the fine structure of the vitelline membrane of the hen's egg and showed it to be up to 12 fL thick and made up of an inner and an outer layer, both fibrous. The inner layer averaged 2·7 fL in thickness (1·0-3·5 fL) and was separated from the outer layer, 3 ·0-8·5 fL thick, by the "continuous membrane" (500-1,000 A). The inner layer, laid down in the ovary, was a three-dimensional network of fibres running mainly parallel to the yolk surface, whereas the outer layer, laid down in the oviduct, consisted of a varying number of sublayers made up of a latticework of fibrils (100 A in their thinnest region). Earlier work based on staining reactions (Moran and Hale 1936 ; McNally 1943; Doran and Mueller 1961) indicated that the inner layer consisted of collagenous material and that the outer layer consisted of mucin, but it is now established that the inner layer is made up of a secreted form of non-collagenous structural connective tissue protein and that the outer layer is made up of proteins similar to those in egg white (Bellairs, Harkness, and Harkness 1963). The vitelline membrane was completely formed in all the developing eggs exa­ mined by Bellairs, Harkness, and Harkness (1963) and, as they were taken either from the lower part of the magnum or the isthmus of the uterus, it was concluded that the outer layer of the membrane must be laid down either in the upper part of the magnum or in the infundibulum of the oviduct. The developing egg spends a very short time in the infundibulum of the oviduct, only 18 min according to Warren and Scott (1935), so chances of finding this stage of development in a sacrificed bird are small. During investigations with White Leghorn hens one such egg was found in the infundibulum and this was used in an

* Division of Food Preservation, CSIRO, P.O. Box 43, Ryde, N.S.W. 2112.

Aust. J. biol. Sci., 1969,22, 653-65 654 JOAN M. BAIN AND JANICE M. HALL

attempt to follow the early stages in formation of the outer layer of the vitelline membrane. These observations, though limited to one egg, clearly relate the secretory processes in the infundibulum with the formation of the outer layer of the vitelline membrane as possibly suggested by Bellairs, Harkness, and Harkness (1963). The structure of the vitelline membranes in ova which had just been liberated from the ovary, but had not yet entered the infundibulum, and vitelline membranes from newly laid eggs were re-examined for comparison. The supposed barrier to diffusion between the yolk and the white of a hen's egg has long been called the vitelline membrane (Liebermann 1888; Lacaillon 1910; Needham 1931; McNally 1943; Romanoff and Romanoff 1949; Doran and Mueller 1961; Fromm 1964, 1967; Holder et al. 1968), but this use of the term "membrane" is now confusing. The nomenclature associated with hen's egg formation is changing with the increased study of its fine structure (Bellairs, Harkness, and Harkness 1963; Bellairs 1964, 1965, 1967; Press 1964; Wyburn, Aitken, and Johnston 1965; Wyburn, Johnston, and Aitken 1965). In the present investigation, the yolk membrane has been called the vitelline membrane; the yolk-containing structure in the ovary, the ovum; the tissue sur­ rounding the ovum, the follicle; the ovulated structure, the ovum; and the structure in the oviduct, the developing egg.

II. MATERIALS AND METHODS (a) Structure of the Vitelline Membrane in an Ovum Just Liberated from the Ovary The ova of the hen are contained in stalked globular structures (the follicles) varying from less than 2 mm to approximately 35 mm in diameter, the largest one being ready to ovulate. The follicle splits to release the ovum into the space above the opening of the infundibulum of the oviduct. Such ova were taken from the space above the opening to the infundibulum of a sacrificed White Leghorn hen and their vitelline membranes prepared for electron microscopy. An incision was made in the membrane to release the yolk, any adhering yolk being washed away with distilled water. The separated membrane was then placed over a plastic ring to keep it fiat and the whole structure was placed in 1 % OsO. in veronal acetate buffer (pH 7·3) for 2! hr. After washing, the material was stained in 2% uranyl acetate and dehydrated in an alcohol series. Small pieces were then cut from the membrane, embedded in Araldite, sectioned, and examined in a Siemens Elmiskop 1 electron microscope at 80 kY. Some sections were stained with lead citrate (Reynolds 1963).

(b) Structure of the Vitelline ~Membrane in a Developing Egg in the Infundibulum The developing egg, found in the infundibulum of the oviduct of a sacrificed White Leghorn hen, stretched from the opening to the entrance ofthe upper part of the magnum (approx. 8·5 cm). The infundibulum, with the enclosed egg, was separated from the oviduct and placed whole in a glass container. A buffered veronal acetate solution with 1 % OsO, (pH 7·3) was poured onto the whole structure and left for 2! hr. Strips of material (infundibulum and underlying egg), approxi­ mately 0·25 cm wide, were then cut at right angles to the long axis of the egg, about 0·5, 1· 5, 3·2, 5·0, 6·7, and 7·7 cm from its upper end to give a developmental sequence; they were fixed for

1 (chalaziferous layer in contact with the white of the egg) and the underlying layer 2. 2 indicates the boundary between layers 2 and 3 and 3 indicates that between layers 3 and 4 (inner layer of the membrane and in contact with the yolk). Width of the membrane is approximately 24 1-'. The continuous membrane (OM) is associated with the outer limit of the inner layer of the vitel­ line membrane. 2·3 X 3,000. VITELLINE MEMBRANE IN HEN'S EGG 655

Figures 1-12 are electron micrographs of material which was fixed in 1 % OsO. and stained with uranyl acetate before embedding. Figures 4-12 were stained with lead citrate on the grid.

Fig. I.-Transverse section of the vitelline membrane from a new-laid egg showing the possible locations where it could be separated into four layers. 1 marks the limit between the outer layer

.------.-.~-.------

VITELLINE MEMBRANE IN HEN'S EGG 657

a further hour in OsO •. Small pieces from each strip were then prepared for electron microscopy as above. During fixation, the under-surface of the infundibulum and the underlying egg material in contact with the glass container were not touched by the fixative. The vitelline membrane from this unfixed area was separated from the egg surface, washed, fixed, and embedded for subsequent sectioning and examination.

(c) Structure of the Vitelline Membrane in a New-laid Egg The shell of the egg was broken soon after laying and the white separated from the yolk. The final separation of the white from the yolk was made by rolling the yolk and the adhering material on a wad of wet filter paper, care being taken not to damage the delicate outer layer of the vitelline membrane. The membrane was separated from the yolk by making an incision in it to allow the yolk to flow away. It was then washed very carefully in several changes of distilled water and could be dissected into four (sometimes five) layers after immersion in distilled water for an hour. The membrane and each of its four separated layers were fixed in 2% KMnO. or 1 % OsO. in veronal acetate buffer (pH 7·3) for 1 hr or 2! hr respectively and then prepared for examin­ ation as above. The delicate material folded easily during this preparation and orientation in the Araldite for subsequent sectioning was difficult. Agar embedding was tried both before and after fixation to keep the membrane flat. Orientation of the material was improved by this method, but the heat of the melted agar altered the structure of the membrane, making it appear similar to that in an incubated egg (Bellairs, Harkness, and Harkness 1963).

III. RESULTS AND DISCUSSION (a) Structure of the Vitelline Membrane in a New-laid Egg A comparison of the thickness of the membranes in individual eggs is difficult because of the delicate nature of the outer layer of the membrane. Although handled carefully during separation from the white and subsequent preparation for electron microscopy, it is uncertain how much of the outer layer may be lost by the time the material is embedded. The stronger fibrous material of the inner layer presents no such problems of separation and measurements of its width in individual eggs are comparable. Values for the thickness of the vitelline membrane vary considerably e.g. 24 fL (Needham 1931), 12-23 fL (Moran and Hale 1936), and 12 fL (Bellairs,

Fig. 2.-Transverse section of vitelline membrane separated from a new-laid egg. The outer chalaziferous layer has been lost from the membrane during preparation for electron microscopy. Loss of this layer is responsible for the wide variation quoted for membrane width. This membrane is approximately 12 fL thick. 2 indicates the limit between layers 2 and 3. 3 indicates the limit between layers 3 and 4. The continuous membrane (OM) is evident on the outer limit of layer 4. 1·5 X 5,000.

Fig. 3.-Transverse section of vitelline membrane separated from an ovum, which had ovulated, but not yet entered the oviduct. At this stage the vitelline membrane consists only of the layer found on the inside of the fully developed membrane. No continuous membrane is present. Some yolk (Y) is adhering to the inside of the vitelline membrane. 1·5 X 10,000.

Fig. 4.-Transverse section of the innermost layer of the infundibulum (0· 5 cm from upper end of egg) showing secreted material (SM) accumulated in a fold of the infundibulum (1) and in close contact with the cilia (0) developed from the epithelial cells. 1·4 X 5,000.

Fig. 5.-Detail of the material (SM) considered to have been secreted by the epithelial cells of the infundibulum (1· 5 cm from the upper end of the egg) and of the cilia (0) developed from them. Occasionally two or more cilia share a common boundary. Transverse section. 1· 8 X 40,000. 658 JOAN M. BAIN AND JANICE M. HALL

Harkness, and Harkness 1963). The presence or absence of the fibrous outermost chalaziferous layer seems to determine this variation. This layer becomes brittle and flakes during dehydration, making it difficult to observe in light or electron microscopy (Fromm 1967). Variation in width of the membrane encountered in the present investigation is shown in Figures 1 and 2. The membrane thickness in Figure 1 is approximately 24 fL' possibly including the chalaziferous layer, whereas the mem­ brane in Figure 2 is approximately 14·5 fL wide. The structure in Figure 2 is compar­ able to that shown in Figure 5 of the paper by Bellairs, Harkness, and Harkness (1963), the chalaziferous layer apparently being absent in this material. Separation of the whole membrane into the inner and the outer layer is relatively easy using a dis­ secting microscope, if the membrane is washed for about an hour in distilled water. Bellairs, Harkness, and Harkness (1963) separated the vitelline membrane into two layers for chemical analysis, after allowing the saline-washed material to dry out on the bottom of a Petri dish. They stated that the "continuous membrane" remains attached to the outer layer formed in the oviduct, when the layers are separated. In the present investigation, when the layers were separated in a wet state, the continuous membrane was usually attached to the inner layer of the membrane. The four layers dissected from the vitelline membrane were distinctive. The outermost or chalaziferous layer was gelatinous (layer 1) with the underlying layer appearing white (layer 2). Beneath this was another white layer (layer 3), whilst the innermost layer (layer 4), next to the yolk, was tough and transparent. The likely limits of these four layers are indicated in Figures 1 and 2. Examination of the struc­ ture of the four layers separated from a membrane indicated that this demarcation is correct. 'Vhen five layers were separated, the extra layer came from a further division of layer 3. Bellairs (1964) points out that there has been disagreement on the number of layers present in the vitelline membrane following investigations with the optical microscope (e.g. Moran and Hale 1936; McNally 1943) and that these interpretations were possible because of the tendency of the outer layer to split haphazardly.

(b) Structure of the Vitelline Membrane nt Ovulation and its Development in the Ovnry The early work on the formation of the vitelline membrane has been reviewed by Romanoff and Romanoff (1949); see also McNally (1943) and Doran and Mueller (1961). The vitelline membrane was thought to appear before the follicle reached 6mm diameter and to be synonymous with the zona radiata. It was thought that the outer layer was formed of intercellular cement from the follicle and that the inner layer was formed from cytoplasm of the oocyte (ovum). In 1963, Bellairs, Harkness, and Harkness showed that only the inner layer was laid down in the ovary. Later it was suggested that the vitelline membrane was secreted by the granulosa cells of the follicle and that it developed between these cells and the zona radiata, which is produced by the membrane of the oocyte (Bellairs 1965). Detailed investigations of the relationship on the inner layer (1 L) of the membrane. The infundibulum cells are considered to be of two types -secretory cells (SO) and cells which have developed cilia (OS). Secreted material not yet incor­ porated in the outer layer of the vitelline membrane lies between these cells and the membrane. The egg yolk material (Y) is shown inside the vitelline membrane. 3 X 2,000. VITELLINE MEMBRANE IN HEN'S EGG 659

Fig. 6.- Transverse section of the epithelial cells of the infundibulum (6·7 cm from the upper end of the egg), the vitelline membrane, and the underlying yolk in the developing egg. The outer layer of the vitelline membrane (OL) is shown adjacent to the infundibulum and developing 660 JOAN M. BAIN AND JANICE M. HALL

Fig. 7.-Further detail of the innermost cells of the infundibulum shown in Figure 6. Two secretory cells (SO) are adjacent to a cell which has developed cilia (OS). 1· 5 X 10,000. Fig. S.-Granular material in the secretory cells of the epithelium of the infundibulum which is probably destined to become part of the outermost layer of the vitelline membrane. 2 X 30,000. VITELLINE MEMBRANE IN HEN'S EGG 661 between follicle cells and the oocyte (ovum), the development of the zona radiata from the oocyte membrane, and the formation of the inner layer of the vitelline membrane between the follicle cells and the zona radiata have been carried out by Press (1964), Bellairs (1967), and Wyburn, Aitken, and Johnston (1965). The developing membrane is first evident in oocytes 2·5 mm in diameter (Bellairs 1965) when it is made up of fibrillae, approximately 100 A in diameter. It does not attain its characteristic appearance until the oocyte is about 8 mm across. The maximum width is reached when the oocyte is approximately 25 mm in diameter, but ovulation does not occur till the oocyte is approximately 35 mm in diameter. The vitelline membrane is now made up of the three-dimensional network of non-collagenous structural connective tissue protein (Bellairs, Harkness, and Harkness 1963) which gives mechanical stability following ovulation. The spaces between the network are filled with a fine granular matrix. Additional pieces of material, possibly projections torn off from the granulosa cells of the follicle, are frequently observed within the network. The structure of the vitelline membrane surrounding an ovum, which had just been liberated, but not yet entered the oviduct, is shown in Figure 3.

(c) Structure of the Vitelline Membrane in a Developing Egg in the Infundibulum Although strips of the infundibulum plus underlying egg material were cut away along the length of the egg to obtain a developmental sequence, the vitelline membrane was found only in close association with the oviduct tissue and the under­ lying yolk in samples taken at an approximate distance of 5·0 and 6·7 cm from the entrance to the upper end. In other samples, taken approximately 0·5, 1· 5, 3·2, and 7·7 cm from this end, only the infundibulum could be identified, the egg material apparently having separated from it during preparation for electron microscopy. The secretory phenomena occurring in the oviduct of the fowl have been investi­ gated by Richardson (1935) and the fine structure observed in the present electron micrographs ofthe cells of the infundibulum is considered to reflect such a process. The infundibulum tissue had numerous folds close to its opening (0·5 cm from the upper end of the egg) with material accumulated in them; the epithelial cells were either ciliated or contained spherical deposits of dense material (Fig. 4). Similar cell structure was found at the 1· 5-cm level, detail of the cilia and the material, possibly secreted by the epithelial cells, being shown in Figure 5. The close association of the cells of the infundibulum and the developing outer layer of the vitelline membrane is shown in Figure 6 (approximately 6·7 cm from the upper end ofthe egg). At this stage, the inner layer is shown to be approximately 2 f.L thick and the outer layer 1·5 f.L thick. The epithelial cells of the infundibulum are elongated and are of two types, which mayor may not alternate with each other. The first type (considered to be a secretory cell) contains conspicuous granular deposits and the second produces the cilia; greater detail of these cells is shown in Figures

Fig. 9.-Transverse section of the vitelline membrane as in Figure 6, showing greater detail of its structure. The outer layer (OL) is shown adjacent to the cilia (0) of the epithelial cells of the infundibulum. The continuous membrane (OM) is evident at the junction of the inner (IL) and outer layers (OL) of the vitelline membrane. 3 X 10,000.

VITELLINE MEMBRANE IN HEN'S EGG 663

7 and 8. Greater detail of the vitelline membrane shown in Figure 6 appears in Figure 9. The outer layer in this figure appears granular' and fairly homogeneous and its structure resembles the granular material in the secretory cells (Fig. 8). Figure 10 shows a vitelline membrane, which has become separated from the infundibulum and underlying yolk during preparation; it gives some evidence of stratification in the outer layer (approximately 6·7 cm from the upper end of the egg), the outer layer appearing as though it could be separating into the morphological layers found in the laid egg. The central area appeared to have a higher proportion of very dense material than the area on either side of it. The entire width of the vitelline membrane is included in Figure 10, two cilia from the infundibulum being present on the outer edge. The membrane was then 5 Jl- wide; the outer layer showed expansion in the freed state (cf. Fig. 9). It was not certa-in whether the outer layer was completely laid down in the infundibulum, as the vitelline membrane was not available for examination at the lowest limit in the structure, or whether further development would take place in the upper part of the magnum. Examination of the membrane separated from the infundibulum and the yolk before fixation (exact location in the infundibulum was not known, but its width shows that it must have been towards the lower end) showed the outer layer to be separating out and to be approximately 7·2 Jl- thick, making the total width of the membrane approximately 9·0 Jl- (Fig. ll). Although the struc­ ture of this outer layer was still rather compact, considerable separation and change of structure could be possible, so as to reach the width and form of the outer layer of the membrane separated from the new-laid egg (14·5-24 Jl-). The earliest stage observed in the formation of the outer layer (approximately 5·0 cm from upper end of egg) is shown in Figure 12. It appeared to be made up of a granular matrix, similar in form to material shown to be secreted by the infundibulum cells, and areas of denser material, which appeared to have formed with condensation of the granules. The continuous membrane appeared to be in a similar form and was obvious at the junction of the inner and the outer layer of the membrane. The two layers were approximately 2·5 and 1 ·25 Jl- wide at this stage. The developing egg was clamped in position in the infundibulum as soon as it was found in the dissected bird, but the question arises whether the positioning of

Fig. 10.-Transverse section of part of the vitelline membrane which has become separated from the infundibulum during preparation for electron microscopy. This membrane is from the same source as that in Figures 6 and 9, but is wider (5 p.) due to separation of the material in the outer layer (OL). There is some evidence of stratification shown. The presence of two cilia (0) from the infundibulum cells indicates that the membrane shown is intact. 1· 7 X 10,000.

Fig. H.-Transverse section of the vitelline membrane, separated from the egg developing in the infundibulum before fixation, showing further expansion of its outer layer than in Figure 10. This membrane is 9 p. in width, but stjll much narrower t~an that~ the laid egg. 1· 4 X 4,000. Fig. 12.-Transverse section of the vitelline membrane in an egg developing in the infundibulum (5·0 cm from the upper end of the egg). The outer layer (OL) of the vitelline membrane is made up of granular material, similar in appearance to that considered to have been secreted by the infundibulum cells (Fig. 8) with areas of greater density developed within it. The continuous membrane (OM) on the surface of the inner layer (IL) appears to be formed from concentrated granules. A cilium (0) from the infundibulum cells is evident. 3·0 X 20,000. 664 JOAN M. BAIN AND JANICE M. HALL the developing egg in the infundibulum was the natural position the egg had reached in the oviduct at the time the bird was killed. It is possible that the egg could have been further down in the oviduct at the time of killing, its position altering durihg this process. The evidence given in Figure 6, however, shows a very close relationship between the secretory activity of the infundibulum and the developing outer layer of the vitelline membrane and seems to indicate that the location of the egg in the infundibulum was a real one. The developmental sequence shown in Figures 6 and 12 supports this conclusion further.

IV. ACKNOWLEDGMENTS The authors are indebted to Dr. L. W. Bobr, Division of Animal Genetics, CSIRO, for providing some of the ova which had just been liberated and the egg developing in the infundibulum of the hen's oviduct, and to Mrs. S. Stephens for technical assistance during the course of the electron microscopy.

V. REFERENCES BELLAIRS, R. (1964).-Biological aspects of the yolk of the hen's egg. In "Advances in Morpho­ genesis". (Eds. M. Abercrombie and J. Brachet.) Vol. 4, pp. 217-72. (Academic Press, Inc.: New York.) BELLAIRS, R. (1965).-The relationship between oocyte and follicle in the hen's ovary as shown by electron microscopy. J. Embryol. expo Morph. 13, 215--33. BELLAIRS, R. (1967).-Aspects of the development of yolk spheres in the hen's oocyte, studied by electron microscopy. J. Embryol. expo Morph. 17, 267-8l. BELLAIRS, R., HARKNESS, M., and HARKNESS, R. D. (1963).-The vitelline membrane of the hen's egg: a chemical and electron microscopical study. J. Ultra8truct. Res. 8, 339--59. DORAN, M., and MUELLER, W. J. (1961).-The development and structure of the vitelline mem­ brane and their relationship to yolk mottling. Poult. Sci. 40, 474--8. FROMM, D. (1964).-Strength distribution, weight, and some histological aspects of the vitelline membrane of the hen's egg yolk. Poult. Sci. 43, 1240-7. FROMM, D. (1967).-Some physical and chemical changes in the vitelline membrane of the hen's egg during storage. J. Fd Sci. 32, 52-6. ' HOLDER, D. P., NEWELL, G. W., BERRY, J. G., and MORRISON, R. D. (1968).-The effect of rations on vitelline membrane strength. Poult. Sci. 47, 326-9. LACAILLON, M. A. (191O).-Sur la structure et la signification de la membrane qui enveloppe la sphere vitelline de l'oeuf des oiseaux. O. r. hebd. Seanc. Acad. Sci., Pari8 150, 240--2. LIEBERMANN, L. (1888).-Ueber einige wenige bekannte Bestandtheile des Hiihnereies. Pjluuer8 Arch. ges. PhY8iol. 43, 71-100. McNALLY, E. H. (1943).-The origin and structure of the vitelline membrane of the domestic fowl's egg. Poult. Sci. 22, 40-3. MORAN, T., and HALE, H. P. (1936).-Physics of the hen's egg. 1. Membranes in the egg. J. expo Bioi. 13, 35-40. NEEDHAM, J. (1931).-The relations between yolk and white in the hen's egg. V. The osmotic properties of the isolated vitelline membrane. J. expo Bioi. 8, 330--44. PRESS, N. (1964).-An unusual organelle in avian ovaries. J. Ultrastruct. Re8. 10, 528-46. REYNOLDS, E. S. (1963).-The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Oell Bioi. 17, 208-12. RICHARDSON, K. C. (1935).-The secretory phenomena in the oviduct of the fowl, including the process of shell formation examined by the microincinerator technique. Phil. Trans. R. Soc. B 225, 149--95. ROMANOFF, A. L., and ROMANOFF, A. J. (1949).-"The Avian Egg." (John Wiley & Sons, Inc.: New York.) VITELLINE MEMBRANE IN HEN'S EGG 665

WARREN, D. C., and SCOTT, H. M. (1935).-The time factor in egg formation. Poult. Sci. 14, 195-207. WYBURN, G. M., AITKEN, R. N. C., and JOHNSTON, H. S. (1965).-The ultrastructure of the zona radiata of the ovarian follicle of the domestic fowl. J. Anat. 99, 469-84. WYBURN, G. M., JOHNSTON, H. S., and AITKEN, R. N. C. (1965).-Specialized plasma membranes in the preovulatory follicle of the fowl. Z. Zellforsch. mikrosk. Anat. 68, 70-9 .

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