Vitelline membrane

Physical Properties. The egg is composed of three basic parts shell, whites (albumen), and yolk. Each of these components has its own membranes to keep the component intact and separate from the other components. The vitelline membrane surrounds the yolk, which in turn is surrounded by the chala2iferous layer of albumen, keeping the yolk in place. Egg white (albumen) consists of an outer thin layer next to the shell, an outer thick layer near the shell, an inner thin layer, and finally, an inner thick layer next to the yolk. Thick layers of albumen have a higher level of ovomucin in addition to natural proportions of all the other egg white proteins. This ovomucin breaks into shorter fibers when the egg white is blended on a high speed mixer (3), or when the egg white ages. Viscosity is gready reduced when the egg white is blended in this way. 

The yolk is separated from the white by the vitelline membrane, and is made up of layers that can be seen upon careful examination. Egg yolk is a complex mixture of water, Hpids, and proteias. Lipid components iaclude glycerides, 66.2% phosphoUpids, 29.6% and cholesterol [57-88-5] 4.2%. The phosphohpids consist of 73% lecithin [8002 3-5] 15% cephahn [3681-36-7], and 12% other phosphohpids. Of the fatty acids, 33% are saturated and 67% unsaturated, including 42% oleic acid [112-80-1] and 7% linoleic acid [60-33-3]. Fatty acids can be changed by modifying fatty acids ia the laying feed (see... 

The isolated oocytes are still surrounded by the vitelline membrane, an inner glycoprotein matrix layer (Yao et al., 2000). This layer does not interfere with whole-cell recording experiments. It can be removed manually however, for patch-clamp experiments requiring the formation of high-resistance seals. 

Aligned prism Vitelline membrane outer layer protein I, subunit A... 

Nelson, D. R. and Leopold, R. A. (2003). Composition of the surface hydrocarbons from the vitelline membranes of dipteran embryos. Comp. Biochem. Physiol. B, 136, 295-308. 

Jones (36) reviewed the natural antinutrients of cottonseed protein products— gossypol and the cyclopropenoic fatty acids (CPFA malvalic and sterculic acids). The CPFAs participate in forming the pink color complex in the Halphen reaction, a test specific for the admixture of cottonseed oil with other oils and fats. They also inhibit A9 desamrase, an enzyme that converts stearic acid into oleic acid, and thus increase hardness of fats from animals (e.g., pig backfat and lard) raised or finished on feedstuffs containing high levels of polyunsamrated oils like corn. Feed industry practice is to limit cottonseed lipids to no more than 0.1-0.2% in the diet of laying hens to avoid pink discoloration of egg whites and alterations of the vitelline membrane that cause pasty yolks. 

Drosophila embryos are protected both by an outer layer called chorion and an impermeable and opaque vitelline membrane. Therefore preparation of whole mount Drosophila embryos for staining with antibodies and/or other fluorescent markers must go through the following steps chorion removal, fixation, vitelline membrane removal, and membrane permeabilization. The next subsection introduces the basic procedures for embryo collection and chorion removal that are common to all protocols described here, as well as the two most common fixation methods with or without methanol (the latter requiring hand devitellinization of embryos). The first one works well for immunostaining, while the second is ideal for F-actin staining with phalloidin. 

Remove the vitelline membrane by hand, individually, under a dissecting microscope using an odontological needle, by making a small hole in the vitelline membrane at one end of the embryo (see Note 6). 

Completely remove the lower (fixative) layer. Add 1 mL methanol and immediately mix it vigorously for about 15 s using a vortex mixer. As the phases reform, devitellinized embryos will sink and stay at the bottom of the tube. Remove the upper and lower layers (in this order), together with embryos that did not lose their vitelline membrane and remain floating between the two phases (see Note 7). 

Electroporation. Whole embryo electroporation in vitro was performed as previously described.1 For targeted regional electroporation (TREP) chick embryos were stained (neutral red) and staged according to Hamburger and Hamilton (HH).3 The vitelline membrane overlying the embryo was removed and a small slit was cut on the yolk membrane near the heart. Platinum electrodes (0.3 mm diameter, at 2.5 mm distance) were mounted on a micromanipulator and positioned parallel to the embryo. 

When the alcohol has burned off, a very hot flame (Tirrell burner) is directed at the drop of water on the under side of the egg and.after sufficient heating a piece of the egg shell from 1 to 2 cm. in diameter snaps off. In some cases the vitelline membrane is broken at this point and the contents of the egg run out, so it is necessary to have a container ready for use. 

If the vitelline membrane does not break at this point or all the contents do not run out, it is only necessary to apply the flame gently to the top (small) end of the egg when the expansion of the air will... 

Two protocols are provided for fixation of Drosophila embryos. Because these embryos are surrounded by both a chorion (eggshell) and an impermeable vitelline membrane, they require special steps to remove these barriers prior to immunostaining or in situ hybridization. Typically, the chorion is dissolved by treatment with sodium hypochlorite (commercial bleach) and the vitelline membrane is removed, either concomitant with or after fixation, by a combination of temperature and osmotic shock. The heptane in these procedures permits the fixative to pass through the vitelline membrane. 

Fig. 2.39 The schematic structure of an egg I eggshell, 2 outer membrane, 3 inner membrane, 4 Chalaza, 5 exterior albumen (outer thin albumen), 6 middle albumen (inner thick albumen), 7 vitelline membrane, 8 nucleus of pander, 9 germinal disk (blastoderm), 10 yellow yolk, 11 white yolk, 12 internal albumen, 13 chalaza, 14 air cell, 15 Cuticle. (Copyright-free Wikipedia picture)...

Lck, a 56-kDa protein, has originally been characterized as a Src-related tyrosine kinase that is specifically expressed in lymphocytes (Lck is named after lymphocyte kinase). In T-cells, Lck associates with CD4/CD8 cell surface receptor for major histocompatibility complex and, upon interaction with antigen-presenting cells, it will be activated by dephosphorylation in the carboxyl-terminal tyrosine residue, as catalyzed by CD45 phosphatase. In murine eggs, it has been reported that CD4-hke structures on the vitelline membranes are involved in gamete interaction, and that Lck-hke protein could have been detected in association with those CD4-like structures (Mori et al. 2000 Mori et al. 1991). While these studies have been done with the use of sjjedfic monoclonal antibodies (e.g. immunofluorescent and immunochemical approaches), biochemical and molecular biological identifications have not yet been demonstrated. 

Mori T, Wu GM, Mori E. 1991. Expression of CD4-like structure on murine egg vitelline membrane and its signal transductive roles through p561ck in fertilization. Am J Reprod Immunol 26(3) 97-103. 

Fig. 7. Fine structures in the egg and around the ovary revealed by transmission electron microscopy, (a) Adjacent pronuclei in an egg filled with yolk granules. Bar = 10 pm. (b) Adjacent part of the pronuclei in (a). Bar = 5 pm. (c) The viteUine membrane (indicated by seven arrows). The envelope is still not completed, but interrupted. The vitelline membrane separates the egg with yolk granules (below) from the lyrate organ (above). Bar = 5 pm. (d) Ovary with several oocytes. Presumed sperms are visible around the ovary. Bar = 5 pm. (e) Presumed sperms between an oocyte and lyrate organ. Bar = 5 pm. (Q Detailed structure of a presumed sperm of (e). The detailed structure in the sperm cell is different from that shown in Di Palma Alberti (2001). Black bars are not a staining artifact but an unknown structure. Bar = 1 pm. Abbreviations Nl, N2, nuclei O, oocyte S, presmned sperm Yl, tipid-yolk granule Yp, protein-yolk granule (unpubHshed micrographs by Toyoshima Alberti).

Fig. 1. Full-grown oocyte within its ovarian follicle. The follicle consists of the outer surface epithelium (SE), the middle theca (T), and the inner follicular epithelium, which, in this particular case, is represented almost entirely by the follicle cell nucleus (FCN). Macrovilli (MAV) from the follicle cell and microvilli (MIV) from the oocyte extend into the substance of the vitelline membrane (VM). Also shown are cortical granules (CG), yolk platelets (YP), pigment granules (PG), and lipid droplets (L). See Smith et al. (1968) for additional information.

The structure of egg yolk within the enclosing vitelline membrane is complex - white, pale yellow and deep yellow layers having been described. The yolk appears to contain a few spheres (of unknown constitution) plus many smaller drops, usually referred to as granules , which are dispersed in a clear yellow medium called plasma. The granules are said to contain 70% of lipovitellins. Both the granules and the plasma contain low-density lipoproteins (LDLs), a proportion of which is based on phospholipids. 

Fertilization occurs during the time between release of the oocyte and its entry into the end of the oviduct. Sperm penetrate the follicle-derived vitelline membrane to fertilize the oocyte and the second reduction division occurs. The resultant one-cell stage embryo is called a blastodisc. The requirement for rapid fertiUzation following follicle rupture is met hy the female chicken s ability to store sperm in viable form for a number of weeks. 

Peristaltic movements carry the egg down the oviduct, a journey that takes about 22 h and during which the egg is subject to a number of modifications. First, a thickened outer layer is applied to the vitelline membrane, which has two extensions, chalazae, which act as stabilizers for the yolk. Albumen is then applied to the outer surface providing a source of water, protein, and antibiotic agents. Next, the double-layered shell membrane is applied, the layers of which are closely apposed to one another, except at the blunt end of the egg, where the gap between them eventually becomes the air space. Finally, in the shell gland, calcite crystals are deposited in and over the outer shell membrane layer to form the eggshell. 

Wash in a dish of Howard s Ringer solution to remove the adherent yolk, cut the embryo from the window, peel away the overlying vitelline membrane, and transfer the embryo to fix. 

Stain young stage embryos (pre stage 15) by applying a drop of neutral red (1% v/v aqueous solution) from the tip of a fine glass rod. The stain will rapidly permeate the vitelline membrane and stain the blastoderm beneath. Stage the embryo (2-4). 

Pull the embryo (plus overlying vitelline membrane) away with a 5 watchmaker s forceps to a clear region of the bowl, and lift by immersing a 30-mm petri dish beneath it and withdrawing it slowly. 

Grip the area opaca with a pair of forceps, and gently shake the embryo free of the vitelline membrane and adherent yolk. 

Gallera and Nicolet (4) tried a more fluid culture medium and were slightly more successful. They also tried a modification of New Culture with a doublering setup in which the vitelline membrane was sandwiched between the two rings, one of which was just small enough to fit into the larger one. This was to facilitate operations to the dorsal aspect of the embryo. The explanted embryo could be turned over without slipping off. 

At the time of laying, the chick embryo is a flat disk consisting of about 60,000 cells. It is made up of two layers, the hypoblast ventrally, lying in close association with the yolk, and the epiblast dorsally, facing the vitelline membrane, the membrane which surrounds the yolk. The disk has a translucent iimer core, the area pellucida, from which the embryo will form, and a denser outer ring of mainly yolk-containing cells, the area opaca. Only the cells at the periphery of the blastoderm are attached to the vitelline membrane, and as they migrate radially, the blastoderm expands. 

Fig. 2. Cartoon of an egg yolk being cut around the equator to remove the vitelline membrane.

When cut right around, take both pairs of watchmaker s forceps and hft vitelline membrane hghtly at the cut edge. Position yolk so that blastoderm is on top, facing the operator. 

Hold the edge of the vitelhne membrane with both pairs of forceps, and pull very slowly by inverting it over the yolk, keeping the membrane low over the yolk, and pulhng towards the bottom of the dish (see Note 7). Stage 4 and 5 blastoderms adhere fairly well to the vitelline membrane and are easy to handle. Older and younger blastoderms need more care. 

When the vitelline membrane is freed from the yolk and the blastoderm stiU attached to it, immerse a watch glass into the BSS and pull the membrane onto it (see Note 8). As you raise the watch glass above the BSS level, hold onto the membrane with tweezers or it will float off. 

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