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.