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Ultrastructural differentiations in the developing follicle cortex of Locusta migratoria, with special reference to vitelline membrane formation.

Electron microscopic studies on developing follicles of Locusta migratoria show the vitelline membrane to be composed of two ultrastructurally distinguishable components: The vitelline membrane bodies (VMBs) and, in addition, fine granular material, cementing the VMBs together. VMBs form first in the oocyte-near zone within the oocyte-follicle cell space. Subsequently, the second vitelline membrane substance is secreted between the VMBs through apical protrusions of the follicle cells. The possible origin of the VMBs is discussed. Yolk uptake in Locusta seems to occur predominantly by pinocytosis. During oocyte development the oocyte membrane is enlarged by numerous microvilli and folds. In addition pinocytotic vesicles are pinched off. It is supposed that the latter loose their coat and eventually transform into large proteid yolk spheres.

Animals

Contributions to an analysis of the avian vitelline membrane's potential to promote outgrowth of the yolk sac-serosal membrane.

Explanted blastoderms of freshly laid chicken eggs expand their area during the first 44-45 hours of incubation by a factor of at least 11 if they are placed with the epiblast on the inner surface of explanted fresh chick vitelline membrane and provided with chick egg extract. This expansion is due essentially to the spreading of the yolk sac-serosal membrane. On turkey and duck membrane the expansion factor is about 6 and 3.8 respectively under otherwise identical conditions, but 1.9 only on a semisolid nutrient agar plate. Only the inner surface of the vitelline membrane has this growth-promoting potential, which markedly and progressively declines during incubation in ovo because of systemic factors rather than because of a direct influence by the outgrowing yolk sac-serosal membrane. Trypsinization of fresh chick vitelline membrane (1% trypsin 3 hours) reduces the growth-promoting potential to about 40% of its normal strength. The outgrowth of the extraembryonic tissues on vitelline membrane is better supported in the presence of a species' own egg extract than by extract from another species.

Animals

The passage of spermatozoa through the vitelline membrane in the domestic fowl, Gallus gallus.

The developing outer layer of the vitelline membrane of the ovum in the posterior part of the infundibulum of the domestic fowl contains many spermatozoa in nearly parallel orientation with its inner layer. When the acrosomal region of a spermatozoon approaches or contacts the inner layer, promptly undergoes the acrosome reaction. The outer acrosomal membrane and overlying plasma membrane fuse together and the apical region of the acrosome opens, so that the acrosomal contents are released. Meanwhile the spermatozoon remains a time in contact with the surface of the inner layer, and the network of the inner layer just under the tip of the sperm head begins to be dissolved. This dissolution extends downward forming a tunnel, approximately 9 micrometer in diameter. The spermatozoon then passes through the inner layer obliquely via the central region of the tunnel and arrives at the perivitelline space.

Animals

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.

Acrosin

Macromolecular components of the vitelline membrane of hen's egg. III. Physicochemical properties of glycoprotein II.

A glycoprotein fraction (GP-II) has been isolated from the vitelline membrane of hen's egg and its physicochemical properties clarified. GP-II is composed of polypeptide (92%), neutral sugar (4%), hexosamine (3.3%), and sialic acid (0.6%). The constituent neutral sugars of this glycoprotein are fucose, mannose, and galactose, in a molar ratio of 2:6:5. An interesting feature of the amino acid composition of GP-II is the high proportion of proline. GP-II exists in an aggregated form and is hydrophobic in nature. Upon velocity sedimentation in 0.5% SDS solution, it showed a hypersharp boundary with an apparent sedimentation coefficient of 5.6 S. Reduction of GP-II, however, gave a single component of 3.6 S which seems to be a subunit of GP-II.

Amino Acids

Nucleoside triphosphatase from the hen's egg white and vitelline membrane: purification, properties and relation to similar enzymes from the oviduct.

Hen's egg white and vitelline membrane nucleoside triphosphatases were purified resulting in active soluble subunits with MR 260,000 +/- 10,000. PH optima are divalent cation dependent and situated at pH 6.2 and 8.0 with ATP and at pH 6.15 with ADP as substrate. Ca2+ and Mg2+ are activators. Km and Ki values for Pi and PPi were determined. The enzymes are specific neither for ATP nor for ADP alone. No separation between nucleoside triphosphatase and nucleoside diphosphatase could be achieved. Differences found in their action can be due to differences in organization and properties of the (intermediary) enzyme-substrate complexes. A close relationship exists with homologous enzymes found in oviductal secretory cells and in oviductal secretions.

Adenosine Triphosphatases

Macromolecular components of the vitelline membrane of hen's egg. II. Physicochemical properties of glycoprotein I.

Of the three major macromolecular components of the vitelline membrane of hen's egg, the lowest molecular weight component (previously designated component I) has been studied and its physicochemical properties clarified. The molecular weight of this component is 27,000 and its chemical composition is typical of a glycoprotein, consisting of protein (91%), total hexose (4.4%), hexosamine (glucosamine 2.3%; galactosamine 0.7%), and sialic acid (1.7%). Uronic acid was not found. The molar ratios of the constituent neutral sugars of this glycoprotein (GP-1) are as follows: fucose 3, mannose 5, galactose 5, glucose 1, and xylose 1. The amino acid profile shows a relatively high proportion of hydrophobic amino acids (39%), which may partly account for the insolubility of GP-I in water.

Amino Acids

Scanning electron microscopy of the vitelline membrane of the hen ovum.

Manual removal of the perivitelline layer overlying the animal pole (AP) reveals three morphologically distinct regions of the vitelline membrane (VM). (1) The central germinal region is 600-800 micron in diameter and is densely populated with pleomorphic microvillous projections. (2) The periblastic region, which also exhibits microvillous projections, is 250-550 micron wide and consists of numerous (80-120) lacunae that are 10-60 micron in diameter and up to 20 micron in depth. (3) At the outer periblastic region, the microvillous projections are less numerous. In the vegetal hemisphere, the VM has few projections and occasionally is discontinuous.

Animals

[Studies on the hatching of miracidia of Dicrocoelium dendriticum (author's transl)].

The eggs of Dicrocoelium dendriticum were induced to open by solutions of formic acid and caproic acid (Table 1). The miracidia hatched in O2-free water after the eggs had been dried with N2 or in vacuum. The miracidia were able to live for 3 hours if water contained 20 mM NaCl, 10 mM KCl, and 1 mM CaCl2. Ca++-ions are obviously necessary for the mobility of miracidia. The experimental use of intestinal juice of the Roman snail Helix pomatia gave hatching results which were dependent on the absence of O2 (exposure to N2) and the presence of bacteria with a still unknown function. The dependence on pH seems to be indirect (Abb. 2). Studies on the permeabilities of the egg shell and the embryonic membrane ("vitelline membrane"), the evidence of an oligosaccharide (Abb. 3) liberated from the "spaltraum" (Abb. 1) during egg-opening, and the determination of the osmotic pressure of the hatching process (50% hatching in 1.2--14. Osmols sucrose/1000 ml H2O; Abb. 4) led to the following hypothesis of hatching mechanism: After the activation of the granular gland of the miracidium an enzyme is released into the extra-embryonic "spaltraum". A polysaccharide is digested to an oligosaccharide which cannot permeate the egg shell and the embryonic membrane. The rising osmotic pressure bursts off the operculum.

Animals

Induction of the cortical reaction in hamster eggs by membrane-active agents.

Fusion of capacitated spermatozoa with the vitelline membrane, but not actual penetration, appears to initiate the cortical reaction in hamster eggs. The reaction can be artificially induced by the application of positively charged particles to the vitelline surface, a situation which may normally be prevented by the zona pellucida. Exposure of hamster eggs to neuraminidase, to lectins (concanavalin A and phytohaemagglutinin-P), to a monovalent ionophore (boromycin) and to 1,3-bis(4-chlorocinnamylideneamino)guanidine elicits a cortical granule discharge resulting in a block to fertilization. These agents all appear to act by inducing depolarization of the vitelline membrane.

Animals

The ultrastructure and function of follicle cells in Foucartia squamulata (Herbst) (Curculionidae).

The follicle cells of Foucartia squamulata are involved in the formation of both vitelline membrane and chorion. Precursors for these egg coverings are synthesized by the rough endoplasmic reticulum and condensed within dictyosomes. The vitelline membrane and the chorion appear on the oocyte surface simultaneously, which is an unusual phenomenon for insects. The follicular epithelium has not been found to contribute to vitellogenesis in the species under study.

Animals

Accumulation, nature, and possible functions of the malachite green affinity material in ejaculated human spermatozoa.

An electron microscopic study was conducted on human sperm over an incubation period of 5 to 6 hours in Tyrode's solution at room temperature. Examination of aliquots of the cells, fixed at timed intervals, with glutaraldehyde, malachite green, and postosmication revealed that malachite green affinity material (MGA-M) was barely discernible at first but did accumulate considerably upon standing. Biochemical analysis of MGA-M, which is extractable by glutaraldehyde, revealed that MGA-M is a mixture of extractable phospholipids and of their lyso-derivatives. Some of these substances have fusogenic properties; i.e., they are able to fuse together the membranes of two different cells. The appearance and accumulation of these fusogens occurred during the incubation period of 5 to 6 hours, which was previously shown to be required to capacitate human sperm in vitro. It is probable, therefore, that human sperm, during their initial period of incubation either in vivo or in vitro, not only become capacitated and undergo the acrosome reaction but also develop the fusogenic substance(s) which are necessary for the imminent fusion of their plasma membrane to the vitelline membrane of the mature oocyte.

Cytoplasm

Membrane events of fertilization in the sea urchin.

Four important events of fertilization in the sea urchin are: 1) the acrosome reaction of the sperm, 2) sperm-egg fusion, 3) the cortical reaction of the egg, and 4) the formation of the fertilization coat. The acrosome reaction is triggered by contact of the sperm with the jelly coat, a complex extracellular matrix surrounding the egg. This causes rapid fluxes of ions, fusion of the acrosome membrane with the plasma membrane, and extension of the acrosomal filament. The acrosome membrane inserted into thesperm plasma membrane covers the acrosomal filament and contacts the plasma membrane of the egg to initiate sperm-egg fusion. One consequence of sperm-egg fusion is insertion of the sperm plasma membrane into the egg plasma membrane, producing a mosaic patch. The sperm components inserted persist in development and can be identified by quantitative methods after gastrulation. Another consequence of sperm-egg fusion is the cortical reaction in which thousands of vesicles fuse with the egg surface, thereby adding their membranes to the egg plasma membrane and releasing their contents upon the egg surface. This results in an approximate doubling of the amount of membrane on the egg surface in a few seconds and produces a mosaic topography. The excess surface membrane is accommodated by elongation of egg microvilli. The cortical reaction causes a detachment of the egg glycocalyx or vitelline membrane, and this layer is elevated from the egg surface. Contents of the cortical granules combine with and alter the vitelline membrane by a hardening reaction to produce the fertilization coat. Hardening involves a peroxidase-mediated tyrosine crosslinking, requiring a burst of oxygen consumption by the egg to generate hydrogen peroxide and resulting in chemiluminescence. These events are followed by activation of metabolic processes in the egg and changes which protect the egg against polyspermy.

Acrosome

Disappearance of cortical granules in rat ovum in vivo following fertilization.

Electron microscopical studies of the rat ova were carried out to clarify the pattern of disappearance of cortical granules following fertilization. When the posterior cap of the head of a spermatozoon was attahced to the vitelline membrane, cortical granules located beneath this membrane fused with this membrane to be decomposed or broken. Then their contents were discharged into the perivitelline space. The disappearance of cortical granules seemed to have started in an area around the site of the vitelline membrane to which spermatozoon was attached and spread soon all over the vitellus.

Animals

Oogenesis and egg-shell formation in Aspiculuris tetraptera Schulz (Nematoda: Oxyuroidea).

The ovary of Aspiculuris tetraptera has a prominent terminal cap cell. This is considered to be part of the ovarian epithelium. Oogonia detach from the short rachis and increase in size from 6 to 60 microns; accumulating hyaline granules, shell granules and glycogen. The hyaline granules persist in the eff cytoplasm after shell formation has been completed and are considered to be lipoprotein yolk. The shell granules contribute to the non-chitin fraction of the chitinous layer. A classification of the cytoplasmic inclusions of the nematode oocyte is proposed. Upon fertilization a vitelline membrane is formed which constitutes the vitelline layer of the egg-shell. The chitinous layer is secreted in the perivitelline space, between the vitelline layer and the egg oolemma. Upon completion of chitinous layer synthesis, the egg cytoplasm contracts away from its inner surface. The material of the lipid layer is secreted at the surface of the egg cytoplasm and adheres to the inner surface of the chitinous layer. During secretion of the chitinous and lipid layers by the egg cytoplasm, the uterine cells secrete the unit membrane-like external uterine layer and the crystalline internal uterine layer. A complex system of interconnecting spaces develops in the internal uterine layer. This system is open to the exterior via breaks in the external uterine layer. There is no direct involvement of the uterine cells in the formation of this structure.

Animals