Egg Vitelline Membrane Research Articles

Proteins of the outer layer of the vitelline membrane of hen's eggs

A study has been made of the proteins in the vitelline membrane of hen's eggs before and after mechanical separation into the inner and outer layers. The membranes were dissolved in detergent (sodium dodecyl sulphate) and chromatographic fractions were examined by gel electrophoresis. The separated inner and outer layers were compared by gel electrophoresis. The outer layer contained (i) enzymically active lysozyme (EC 3.2.1.17) (about 60% dry weight), (ii) an insoluble ovomucin complex and (iii) a new protein, VMOI (vitelline membrane outer I). These account for most of the protein. In addition, some minor constituents were detected by gel electrophoresis but were not isolated. Except for ovomucin, the constituents of the outer layer could be dissolved from the membrane at high ionic strength ( > 0.5 M sodium chloride), resulting in a loss of its structure. On lowering the ionic strength the soluble proteins recombined with the membrane, partially regenerating the original structure. Ovomucin appears to form the skeleton of the outer layer, but the salt-soluble proteins, especially lysozyme, are responsible for its integrity. The function of the newly-recognized protein (VMOI) is not known. Its molecular weight is 17500 according to gel electrophoresis in detergent and it contains no methionine. The inner layer consists largely of the proteins GP1, GPII and GPIII isolated by Kido et al. (Kido, S., Janado, M. and Nunoura, H. (1975) J. Biochem. 78, 261–268) from the whole membrane.

Chemical characterisation of the vitelline membrane of hens' eggs

The chemical composition of the vitelline membrane of hens' eggs, both fresh and stored (±1°C) for 6 months, has been studied. The results indicate that the vitelline membrane contains mainly protein compounds with some carbohydrates and lipids. The amounts of particular components depend on the method of preparation. During cold storage there is a loss of nitrogen and changes in the chemical composition of the vitelline membrane should first be considered in respect of the protein part of the membrane.

Analysis of Human Spermatozoal Fertilizing Ability Using Zona-Free Ova

An in vitro fertilization assay employing zona-free hamster eggs was used to analyze human spermatozoal fertilizing ability. Human spermatozoa were preincubated for 18 to 20 hours in Biggers, Whitten, and Whittingham's medium (1971) at a concentration of 1 X 10(7) sperm/ml prior to the addition of zona-free superovulated hamster eggs. Eggs were examined microscopically 2 hours later for evidence of swelling or decondensing sperm heads in the cytoplasm. A total of 6266 eggs were examined in assays for both suspected fertile and infertile donors; 50 eggs/sample were examined. The percentage fertilization was found to range from 14% to 100% in the suspected fertile group with an average of 56.3%. The sperm concentration in this fertile group ranged from 22 to 303 million/ml with an average of 114. The suspected infertile samples yielded fertilization rates of 10% or less and an average count of 50.6 million/ml. These data suggest that human spermatozoa fuse with the vitelline membrane of zona-free hamster eggs and decondense with varying efficiencies. The percentage of fertilization in this cross-species system did not show a significant correlation with sperm concentration or motility. However, suspected infertile samples always yielded 10% or less fertilization in this assay. This method may have potential value as a diagnostic tool in evaluating human spermatozoal fertilizing capacity which avoids the ethical and logistcal problems associated with fertilization of human eggs in vitro.

Hydrolysis of the hen egg vitelline membrane by cock sperm acrosin and other enzymes.

A technique utilizing Pregnant Mare's Serum Gonadotropin and Human Chorionic Gonadotropin treatment of hens (Gallus domesticus), followed by manual ovulation of the excised follicles, was developed to obtain a large number of mature ova. The intact ova were used to test whether acrosin, partially purified from the spermatozoa of the cock (Gallus domesticus), partially purified rabbit testicular acrosin and commercial preparations of several hydrolytic enzymes could dissolve the inner vitelline membrane. Enzymes were applied to pieces of filter paper placed on the ovum. Cock acrosin and endopeptidases such as trypsin, chymotrypsin, collagenase and elastase hydrolyzed the membrane whereas exopeptidases such as leucine aminopeptidase and carboxypeptidase A did not. Phospholipase A, sulfatase, hyaluronidase, beta-glucuronidase and rabbit testicular acrosin also failed to hydrolyze the membrane. Cock acrosin hydrolysis of the ovum surface was inhibited by soybean trypsin inhibitor. The surface of the ovum over the germinal disc region was hydrolyzed more quickly by cock acrosin than the surface over other regions of the ovum. Acrosin from cock sperm caused the release of trichloroacetic acid soluble material absorbing at 280 nm from sonicated preparations of inner vitelline membranes. Hydrolysis was greatest at pH 8.0 and was inhibited by soybean trypsin inhibitor.

Physiological studies on the sperm surface component responsible for sperm-egg bonding in sea urchin fertilization: I. Effect of sperm-binding protein on the fertilizing capacity of sperm

When sea urchin sperm is pretreated with sperm-binding protein prepared from the vitelline membrane of eggs of homologous species, it loses its fertilizing capacity entirely without losing its motility. It is not affected at all by sperm-binding protein from heterologous species. Neither agglutination nor acrosome reaction is evoked by the pretreatment. It is suggested that the sea urchin spermatozoon has on the apical part of its head a component which is complementary to the sperm-binding protein of the egg, and that the observed loss of the fertilizing capacity is caused by antedated interaction of this component with sperm-binding protein added before insemination.

ATPase activities of inner and outer regions of the vitelline membrane during development

ATPase activity has been demonstrated to be present in the vitelline membrane complex of the hen's egg. This activity increases markedly after ovulation. The vitelline membrane complex has been isolated by dissection and studied in an Ussing chamber such that the inner and outer surfaces can be examined independently. Coupled activity has been detected whereby the presence of substrate on one side affects activity on the other side. Measurements demonstrate that the vitelline membranes of eggs laid in the fall have low enzymatic and no coupled activity. The vitelline membranes of eggs laid in the summer have high enzymatic and marked coupled activity. The coupled activity is reversed in the fertilized compared to the unfertilized egg. This study sheds light on both the physiological character of the ATPases and the roles played by the vitelline membrane in the development of the ovum prior to and after fertilization.

Methods for removal of the vitelline membrane of sea urchin eggs: I. Use of dithiothreitol (Cleland Reagent)

A simple procedure is described for removing the vitelline membrane of sea urchin eggs. The method is based on the observation that brief incubation of unfertilized sea urchin eggs in 5–10 mM dithiothreitol (DTT or Cleland Reagent) at pH 9.2 effectively removes this membrane. Membrane removal and normal development have been obtained with the eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus. That DTT removes membranes, combined with other observations and experiments, indicates that the structural integrity of the vitelline membrane results in part from disulfide linkages between neighboring protein molecules.

Methods for removal of the vitelline membrane of sea urchin eggs: II. Controlled exposure to trypsin to eliminate post-fertilization clumping of embryos

A simple modification of the trypsin method for removing the vitelline membrane of sea urchin eggs yields membraneless eggs which do not clump appreciably after insemination. The modification entails minimal incubation in dilute trypsin (0.25 mg%). It is postulated that although this procedure functionally removes the vitelline membrane, patches of membrane remain on the surface and prevent adhesion between hyaline layers of adjacent cells.

Role of the oocyte nucleus in physiological maturation in Rana pipiens

The events of maturation (germinal vesicle dissolution and meiosis to the second metaphase) have been induced with progesterone in vitro in full-grown oocytes dissected from their ovarian follicles. Oocytes from which germinal vesicles (GV) were removed prior to progesterone exposure were subsequently tested for the capacity to be artificially activated, cleave, and synthesize control levels of protein. The enucleated eggs responded to an activation stimulus when tested about 48 hours after a 1-hour exposure to progesterone. This positive response included breakdown of the cortical granules, elevation of the vitelline membrane, rotation within the perivitelline space, increase in egg turgidity, and changes in the appearance of the egg surface. Removal of the egg vitelline membrane and any adherent follicle cells with pronase, prior to progesterone exposure, did not prevent enucleated eggs from undergoing activation; the sole criterion in this case was breakdown of the cortical granules. Many of the enucleated, activated eggs displayed abortive cleavages. Enucleated eggs lacking GV material in their cytoplasm were incapable of genuine cleavage when tested by nuclear transplantation. This restriction was partially removed by injecting GV material back into the enucleated progesterone-stimulated eggs. Donor GV material from non-hormone-treated eggs was essentially as effective in this respect as GV material from progesterone-stimulated eggs. Incubation of oocytes with actinomycin D at concentrations as high as 50 μg/ml had no effect in progesterone-induced maturation. Likewise, injection of actinomycin D directly into oocytes did not prevent progesterone-induced maturation. Puromycin, on the other hand, suppressed all stages of maturation. These results suggest that maturation does not require concomitant DNA-dependent RNA synthesis, but that protein synthesis is obligatory for all stages of maturation. Protein synthesis was measured in eggs enucleated prior to progesterone exposure and at several times after the hormone stimulation. The rate of protein synthesis during the entire period of maturation was essentially identical in enucleated eggs and their nucleated controls. The rate was initially low, increased only after progesterone exposure, reached a maximum level about when nucleated controls gave off their first polar bodies, and the elevated rate was maintained for at least a day after nucleated controls were first capable of being fertilized. Based on these experiments, we conclude that the mixing of GV material with the egg cytoplasm is not a prerequisite to attaining the capacity to undergo artificial activation or to synthesize protein, but is required for the enucleated eggs to cleave. We further suggest that the site of action of progesterone in inducing maturation is extranuclear, possibly at the egg surface.

An egg cell-membrane derivative on the vitelline membrane of annelid eggs

An egg cell-membrane derivative on the vitelline membrane of annelid eggs

THE LIPID COMPONENTS IN THE VITELLINE MEMBRANE OF ASCARIS LUMBRICOIDES EGGS

The preparation of embryonated ascaris eggs enclosed only by the vitelline membrane is described. No impairment of membrane structure or function was observed. Membrane and larval fractions were separated, and their lipids analyzed. The membrane lipids were nearly all unsaponifiable, and consisted mainly of ascaryl alcohol. Larval lipids were primarily saponifiable, but also contained significant amounts of ascaryl alcohol, and nearly all of the sterols. Because the vitelline membrane dissolved in chloroform and similar solvents, ascaryl alcohol was judged to be a major component. The results are compared with previous observations and hypotheses concerning the vitelline membrane of nematode eggs.

THE LIPID COMPONENTS IN THE VITELLINE MEMBRANE OF ASCARIS LUMBRICOIDES EGGS

The preparation of embryonated ascaris eggs enclosed only by the vitelline membrane is described. No impairment of membrane structure or function was observed. Membrane and larval fractions were separated, and their lipids analyzed. The membrane lipids were nearly all unsaponifiable, and consisted mainly of ascaryl alcohol. Larval lipids were primarily saponifiable, but also contained significant amounts of ascaryl alcohol, and nearly all of the sterols. Because the vitelline membrane dissolved in chloroform and similar solvents, ascaryl alcohol was judged to be a major component. The results are compared with previous observations and hypotheses concerning the vitelline membrane of nematode eggs.