Yolk Degradation Research Articles

Regulation of yolk degradation, or how to make sleepy lysosomes

ABSTRACT

Degradation of Red Imported Fire Ant (Hymenoptera: Formicidae) Yolk Spheres

Transmission electron microscopy reveals that the eggs of the red imported fire ant, Solenopsis invicta Buren, contain yolk spheres that are evenly distributed throughout the center of the egg. Yolk degradation occurs sequentially as evidenced by the simultaneous observation of three stages of yolk platelet degradation within the eggs.

Changes in yolk platelet pH during Xenopus laevis development correlate with yolk utilization: A quantitative confocal microscopy study

The variations of the pH in Xenopus yolk platelets have been estimated by fluorescence confocal microscopy and computer image processing. For pH measurements in vitellogenic oocytes, the pH-sensitive fluorescent dye, DM-NERF, was coupled to vitellogenin, and the DM-NERF-vitellogenin was taken up by oocytes via receptor-mediated endocytosis. Dual emission ratio measurements of internalized DM-NERF-vitellogenin indicated that the mature yolk platelets are mildly acidic (pH 5.6). Their precursors, the primordial yolk platelets, have a similar pH. This pH is probably sufficiently low for the partial cleavage of vitellogenin to yolk proteins, but not for yolk degradation. The yolk platelet pH at various developmental stages was estimated by measuring the accumulation of Acridine Orange, both in isolated yolk platelets and in disaggregated embryonic cells. During oogenesis, the yolk platelets accumulated a constant amount of Acridine Orange, corresponding to a pH of around 5.7. During embryogenesis, however, yolk platelets became progressively much more acidic (pH < 5). Acidification correlated with yolk degradation in the various tissues examined, and yolk utilization was blocked when acidification was inhibited with bafilomycin, an inhibitor of vacuolar H+-ATPase. Bafilomycin also inhibited differentiation of cells isolated from stage 13-15 embryos. These data show that the yolk platelet pH is developmentally regulated and is involved in triggering yolk degradation. Also, yolk acidification and degradation appeared to be associated with cell differentiation and with the formation of the endosomal/lysosomal compartment, typical of adult cells, but absent in early embryos.

An Ultrastructural Investigation on Vitellophage Invasion of the Yolk Mass during and after Germ Band Formation in Embryos of the Stick Insect Carausius morosus Br.: (embryogenesis/yolk/vitellophage/Carausius morosus/scanning and transmission electron microscopy).

Developing embryos of the stick insect Carausius morosus were examined ultrastructurally with a view to studying vitellophage invasion of the yolk mass during and after germ band formation. Newly laid eggs in C.morosus have a unique yolk fluid compartment surrounded by a narrow fringe of cytoplasm comprising several small yolk granules. Vitellophages originate mainly from a thin layer of stem cells, the so-called yolk cell membrane, interposed between the germ band and the yolk mass. Throughout development, a thin basal lamina separates the yolk cell membrane from the overlying embryo. Vitellophages extend from the yolk cell membrane with long cytoplasmic processes or filopodia to invade the central yolk mass. Along their route of entrance, filopodia engulf portions of the yolk mass and sequester it into membrane-bounded granules. As this process continues, the yolk mass is gradually partitioned into a number of yolk granules inside the vitellophages. Later in development, the yolk cell membrane is gradually replaced by the endodermal cells that emerge from the anterior and posterior embryonic rudiments. From this stage of development onwards, vitellophages remain attached to the basal lamina through long filopodia extending between the endodermal cells. Yolk confined in different vitellophagic cells appears heterogeneous both in density and texture, suggesting that yolk degradation may be spatially differentiated.

Yolk Degradation in Tick Eggs: III. Developmentally Regulated Acidification of the Yolk Spheres: (Ornithodoros moubata embryogenesis/vitellin/acidic compartment/acridine orange).

Yolk spheres in tick eggs contain a latent procathepsin L, which is activated in vivo, in parallel with yolk degradation, and in vitro by acid treatment (Fagotto, F., Arch. Insect Biochem. Physiol., 1990b in press). Mature cathepsin L hydrolyzes vitellin at acidic pH (Fagotto, F., Arch. Insect Biochem. Physiol., 1990a in press). Here, yolk spheres' pH has been estimated using acridine orange. In the early development, all yolk spheres are neutral, then an increasing number acidify, until hatching, where general acidification seems to occur. This fits well with vitellin utilized slowly during embryogenesis, more intensely at hatching (Chinzei, Y. and I. Yano, Experientia 41, 948, 1985), and can be related to sequential degradation of individual spheres observed during embryonic development, then extensive yolk liquefaction in the larva. Different yolk spheres populations have been separaded on Percoll density gradients. In freshly laid eggs, yolk spheres are dense, neutral, undegrated and contain exclusively the precursor of cathepsin L. In later stages, yolk spheres are progressively recovered in lower density fractions, displaying acidic interior and cytological signs of degradation. They cosediment with mature cathepsin L. It is concluded that acidification initiates yolk degradation through procathepsin L activation.

Yolk degradation in tick eggs: II. Evidence that cathepsin L‐like proteinase is stored as a latent, acid‐activable proenzyme

Cathepsin L-like proteinase found in the eggs of the tick Ornithodoros moubata is latent during embryogenesis, but can be activated by acid treatment. In crude extracts as well as in partially purified fractions, activation requires reducing conditions and is inhibited by leupeptin, which indicates that it is mediated by a thiol proteinase, probably by the cathepsin L itself. Latency disappears in vivo at the time of the acute phase of yolk digestion, which takes place during late embryonic development and larval life. When egg cathepsin L is localized through its gelatinolytic activity on SDS-PAGE, the activated enzyme migrates as lower Mr bands than the latent form. Disappearance of the higher Mr bands corresponding to the latent form is directly related to appearance of the lower Mr bands characteristic of the active one; transition from one pattern to the other and enzymatic activation are in perfect agreement with regard to kinetics and sensitivity to inhibitors. The same pattern change occurs in vivo, parallel to latency removal and intense yolk degradation. These results strongly suggest that egg cathepsin L is stored in the yolk as a proenzyme which is activated by partial proteolysis at low pH.

Ultrastructural Studies on Kronborgia (Platyhelminthes, Fecampiidae) - the Differentiated Vitellocyte of Kronborgia-Isopodicola Blair and Williams

In the adult parasitic female form of K. isopodicola, the vitellocyte cytoplasm contains yolk platelets, lipid bodies, glycogen, myelin figures and mitochondria. The platelets consist of an outer granular zone and an opaque core. All platelets are surrounded by several membranes. Many are cloven into segments by fissures containing membranes. Frequently, small peripheral fragments of the granular zone are pared away, and the core undergoes fragmentation by the same process. Spherical dense bodies are found in the cytoplasm. During the cocoon phase, the platelets are often intricately fragmented, and many pieces are paracrystalline. In the newly deposited egg, many platelets comprise only core segments, which are typically paracrystalline, frequently polygonal, and enveloped by multiple membranes. Spherical dense bodies are not encountered at this stage. The platelets are unlike the 'yolk globules' of Digenea, but are similar to vitelline platelets described for polyclads. In morphology and mode of utilisation they bear some resemblance to yolk granules of Amphibia. The membranes are interpreted as isolating membranes of cellular autophagy. Glycogen synthesis is related to autophagic events involved in yolk degradation. The spherical bodies probably represent eggshell granules; complex shell granules, characteristic of other platyhelminths, were not observed in K. isopodicola.

Yolk degradation in tick eggs: I. Occurrence of a cathepsin L‐like acid proteinase in yolk spheres

In crude extracts of eggs of the soft tick Ornithodoros moubata, maximum degradation of vitellin is at pH 3-3.5, whereas no proteolysis is detected at neutral or weakly acidic pHs. Acidic proteolysis is maintained at high level throughout embryonic development, and rapidly decreases in the larva, during the high phase of yolk degradation. Proteinase, acid phosphatase, and N-acetylglucosaminidase are localized within the yolk spheres; these can be considered as lysosomal-like organelles containing both substrate (vitellin) and the degradative machinery. Proteolytic activity has been essentially attributed to a cathepsin L-like enzyme through substrate specificity and inhibitors. The molecular weight is 37,000 to 39,000 as shown using gelatin-containing SDS-PAGE activity gels. At neutral pH the enzyme binds to vitellin, as demonstrated by gel filtration and PAGE under nondenaturing conditions. Acid proteinase activity at pH 5-6 is undetectable both with proteins and synthetic substrates, but is strongly increased after preincubation at pH 3-4. Activation at low pH could be important in the regulation of yolk degradation.

The Maternal Origin of Acid Hydrolases in Drosophila and Their Relation with Yolk Degradation: (Drosophila/acid hydrolases/developmental regulation/yolk degradation/mitochondria).

The acid hydrolases of Drosophila are of maternal origin and appear subjected to differentiated control during embryogenesis. The enzymes are found associated with yolk granules. This association decreases during embryogenesis, in parallel with yolk degradation. As suggested before (Medina et al. Arch. Biochem. Biophys., 263, 355-363) the acid proteinase seems to be involved in the degradation of the yolk protein. The developmental profile of activity of the proteinase fits rather well with its involvement in the degradation of yolk granules. We have isolated intermediates of degradation of these subcellular structures. The intermediates have acid hydrolase activity and decrease in buoyant density during embryogenesis, in parallel with yolk degradation. The electron microscopic analysis has revealed that they are morphologically heterogenuous. A population of yolk granules appears to store mitochondria in their interior. The mitochondrial marker cytochrome oxidase is detected in density gradients associated with the intermediates of degradation, also supporting the storage of mitochondria in yolk granules in early development. The fact that the acid hydrolases are of maternal origin suggests that they have a role during embryogenesis. We propose that acid hydrolase(s) are involved in yolk degradation.

Mechanisms of yolk degradation in Artemia: A morphological study

1. 1. The electron microscopic analysis of the intermediates of yolk degradation in Artemia has revealed that the process is not homogeneous. Three different mechanisms of degradation (1, 2 and 3) with different complexity appear to occur during development. 2. 2. We have described previously that yolk degradation in Artemia occurs through five cycles. 3. 3. The three mechanisms have not been found in every cycle. 4. 4. Mechanism 2 has been observed in cycle one, two and five, and both mechanisms 1 and 3, in the third and fourth cycles. 5. 5. Mechanism 3 releases mitochondria thus confirming previous results on the storage of mitochondria in the yolk granules of Artemia.

Drosophila cathepsin B-like proteinase: A suggested role in yolk degradation

A cysteine, cathepsin B-like proteinase activity has been found in Drosophila embryos. It appears associated with yolk granules and its activity during embryogenesis correlates well with the degradation of these organelles. In mature oocytes, the enzyme is found in an inactive form which may be activated by limited proteolysis by a serine proteinase also present in oocytes. In early embryos, when solubilized in vitro, the cathepsin B-like proteinase is found in a form of high molecular mass (approx 1000 kDa). This decreases with development down to about 39 kDa, likely the mass of the free proteinase. The heavy form apparently results from the tight association with a yolk protein complex. The proteinase has been found in vitro to degrade readily the yolk polypeptides. The proteinase activity increases during early embryogenesis in parallel with the decrease in molecular weight of the heavy form, and decreases to low values in late embryos. We have also found that ammonium chloride can inhibit in vivo the degradation of yolk and, in parallel, the developmental inactivation of the proteinase. The results altogether suggest that the cathepsin B-like proteinase is implicated in yolk degradation in Drosophila.

Acid hydrolases during Artemia development: A role in yolk degradation

1. 1. A broad group of acid hydrolytic activities have been determined during Artemia development including cathepsin B acid ribonuclease, acid deoxyribonuclease, acid phosphatase, acid phosphodiesterase, β-glucosidase, β- N-acetylgalactosaminidase and acid lipase. These enzymes present maximum activity in nauplii, when yolk degradation is maximum. 2. 2. Artemia cathepsin B proteinase is able to degrade lipovitellin in vitro with a pattern similar to that found in vivo. These results suggest the involvement of acid hydrolases in the degradation of yolk. 3. 3. In cryptobiotic embryos, acid hydrolases were found associated with structures of buoyant density 1.18. The structures appeared at the electron microscope as 0.4 μm dia. vesicles when revealed by acid phosphatase cytochemistry.

Ultrastructure of the pre-implantation shark yolk sac placenta

During ontogeny, the yolk sac of viviparous sharks differentiates into a yolk sac placenta which functions in gas exchange and hematrophic nutrient transport. The pre-implantation yolk sac functions in respiration and yolk absorption. In a 10.0 cm embryo, the yolk sac consists of six layers, viz. (1) somatic ectoderm; (2) somatic mesoderm; (3) extraembryonic coelom; (4) capillaries; (5) endoderm; and (6) yolk syncytium. The epithelial ectoderm is a simple cuboidal epithelium possessing the normal complement of cytoplasmic organelles. The endoplasmic cisternae are dilated and vesicular. The epithelium rests upon a basal lamina below which is a collagenous stroma that contains dense bodies of varying diameter. They have a dense marginal zone, a less dense core, and a dense center. The squamous mesoderm has many pinocytotic caveolae. The capillary endothelium is adjacent to the mesoderm and is delimited by a basal lamina. The endoderm contains yolk degradation vesicles whose contents range from pale to dense. The yolk syncytium contains many morphologically diverse yolk granules in all phases of degradation. Concentric membrane lamellae form around yolk bodies as the main yolk granules begin to be degraded. During degradation, yolk platelets exhibit a vesicular configuration.

Effects of serotonin and serotonin antagonists on chick embryogenesis.

Serotonin and some selected substances known to interfere with its formation (diethyldithiocarbamate) and function (Catron®, 5-methyltryptamine, promethazine) were tested for their ability to affect chick embryo morphogenesis during the first 48 h of development. To detect possible differences in sensitivity between the successive morphogenetic events taking place during this period, the treatment was begun at successively more advanced stages corresponding to embryo ages of between 4 and 30 h incubation. In all cases, the treatment was terminated at an embryo age of 48 h incubation. The treatment was performed both in ovo and in vitro.With some exceptions, the substances induced malformations of the same characteristic types. The developmental processes subjected to disturbances included blastoderm expansion, primitive streak formation, neurulation with brain formation, and somitogenesis. At the cellular level, the malformations can be traced to delayed yolk degradation, impaired formation and function of microvilli, and impaired ability of the embryo cells to change shape.All of the tested chemicals can be expected to interfere with intracellular levels of serotonin. They obviously interfered with decomposition of the yolk granules, recognized centres for intracellular serotonin formation and we therefore conclude that the observed morphogenetical disturbances are ultimately due to impairment of the endogenous serotonin formation. We suggest that, in morphogenesis, serotonin primarily promotes the activity of microtubules and microfilaments.