The effects of pH and weak bases on the in vitro endocytosis of vitellogenin by oocytes of Drosophila melanogaster

Summary The endocytosis of labeled vitellogenin by the developing oocytes of Drosophila melanogaster is pH dependent and inhibited in the presence of primary amines as determined by culturing whole ovaries in vitro. When the pH of the culture medium is adjusted to 6.8 or above, the vitellogenic oocytes sequester labeled vitellogenin synthesized by the follicle cells. The endocytosis of vitellogenin is shown autoradiographically by the accumulation of labeled yolk spheres within the oocytes. When the pH of the medium is reduced to 6.6 or below, the oocytes fail to sequester labeled vitellogenin, as demonstrated by an increase in immunoprecipitable vitellogenin in the culture medium and a concomitant reduction in the number of labeled yolk spheres within the oocytes. Vitellogenin endocytosis is also impaired by the addition of the primary amines methylamine or chloroquine to the culture medium. Monensin, a carboxylic ionophore, is shown to inhibit completely the secretion of labeled vitellogenin from the follicle cells.     

Development of male-incubated ovaries in the gypsy moth,Lymantria dispar

Ovaries from Lymantria dispar females were transplanted into an environment lacking vitellogenin, the male milieu, in order to determine how the presence of vitellogenin in the hemolymph affects the process of protein uptake by gypsy moth oocytes. When undeveloped ovaries from newly ecdysed last instar females were transplanted into males of the same stage, follicles detached from the germarium and increased in size, but the growth of oocytes proceeded more slowly than those from female controls. Although chorion fromation was delayed in male-grown ovaries, scanning electron microscopy of chorionated eggs recovered from adult males showed that a chorion with normal surface architecture was formed by the adult stage. SDS-PAGE analysis of the male-grown ovaries and hemolymph from males receiving ovaries showed that vitellogenin production was not stimulated by the organ transplant and only male hemolymph proteins were internalized by the male-incubated ovaries. Thus, in the absence of vitellogenin, endocytosis of male hemolymph proteins occurred, but the rate of oocyte growth was slowed.     

Lipid uptake by insect oocytes

Approximately 30-40% of the dry weight of an insect egg consists of lipid, mostly triacylglycerol (TAG). Although this lipid is essential for the energy needed by the developing embryo, little is known about the mechanism that leads to the accumulation of TAG in the insect egg. Insect oocytes can readily synthesize TAG from free fatty acids (FFAs) and glycerol, however, de novo synthesis of FAs by the oocyte is marginal. Hence, FAs have to be imported from the fat body or the diet. Insect hemolymph contains two lipoproteins that transport lipids, lipophorin and vitellogenin. Both are taken up via endocytosis by the oocyte, however, this provides only about 10% of the egg's lipid reserves. The rest is unloaded from circulating lipoprotein particles at the oocyte surface in the form of diacylglycerol (DAG), the major lipid transport form in insects, or as FFA. The mechanism of lipoprotein unloading at the oocyte surface is currently unclear. Possible roles of the lipid transfer particle (LTP), FA transporters, and lipoprotein lipase activity are discussed.     

Involvement of the GTP binding protein Rho in constitutive endocytosis in Xenopus laevis oocytes

To study an endocytotic role of the GTP-binding protein RhoA in Xenopus oocytes, we have monitored changes in the surface expression of sodium pumps, the surface area of the oocyte and the uptake of the fluid-phase marker inulin. Xenopus oocytes possess intracellular sodium pumps that are continuously exchanged for surface sodium pumps by constitutive endo- and exocytosis. Injection of Clostridium botulinum C3 exoenzyme, which inactivates Rho by ADP-ribosylation, induced a redistribution of virtually all intracellular sodium pumps to the plasma membrane and increased the surface area of the oocytes. The identical effects were caused by injection of ADP-ribosylated recombinant RhoA into oocytes. The C3 exoenzyme acts by blocking constitutive endocytosis in oocytes, as determined using a mAb to the beta 1 subunit of the mouse sodium pump as a reporter molecule and oocytes expressing heterologous sodium pumps. In contrast, an increase in endocytosis and a decrease in the surface area was induced by injection of recombinant Val14-RhoA protein or Val14-rhoA cRNA. PMA stimulated sodium pump endocytosis, an effect that was blocked by a specific inhibitor of protein kinase C (Go 16) or by ADP-ribosylation of Rho by C3. Similarly, the phorbol ester-induced increase in fluid-phase endocytosis in oocytes was inhibited by Go 16, C3 transferase, or by injection of ADP-ribosylated RhoA. In contrast to C3 transferase, C. botulinum C2 transferase, which ADP-ribosylates actin, had no effect on sodium pump endocytosis or PMA-stimulated fluid- phase endocytosis. The data suggests that RhoA is an essential component of a presumably clathrin-independent endocytic pathway in Xenopus oocytes which can be regulated by protein kinase C.     

An unusual lysosome compartment involved in vitellogenin endocytosis by Xenopus oocytes

We have investigated the lysosomal compartment of Xenopus oocytes to determine the possible role of this organelle in the endocytic pathway of the yolk protein precursor, vitellogenin. Oocytes have lysosome-like organelles of unusual enzymatic composition at all stages of their development, and the amount of hydrolase activity increases steadily throughout oogenesis. These unusual lysosomes appear to be located primarily in a peripheral zone of oocyte cytoplasm. At least two distinct populations of lysosomal organelles can be identified after sucrose density gradient fractionation of vitellogenic oocytes. Most enzyme activity resides in a compartment of large size and high density that appears to be a subpopulation of yolk platelets that are less dense than most platelets within the cell. The appearance of this high density peak of lysosomal enzyme activity coincides with the time of onset of vitellogenin endocytosis during oocyte development. The data suggest that endocytic vesicles that contain vitellogenin fuse with modified lysosomes shortly after their internalization by the oocyte. Pulse-chase experiments with radiolabeled vitellogenin suggest that the ligand passes through the low density platelet compartment en route to the heavy platelets. The accumulation of yolk proteins apparently results from a failure of these molecules to undergo complete digestion after their entry into an unusual lysosomal compartment. The yolk platelets that these proteins finally enter for prolonged storage appear to be a postlysosomal organelle.     

抑卵激素对家蝇卵巢周期性发育的调控

抑卵激素是调控家蝇Musca dorncstica vicina卵巢周期性发育的关键因子之一。在家蝇中,当第一个周期的卵母细胞处于卵黄发生期或卵黄发生后期时,其第二个周期的卵母细胞的发育不进入卵黄发生期。本文建立了家蝇抑卵激素的生物测定方法,即用一对卵巢提取物注射1头羽化后12h家蝇,并在羽化后60h观察卵母细胞的发育及卵黄蛋白的沉积情况。抑卵激素的作用首先是延缓了卵母细胞在卵黄发生前期的发育;其次,抑卵激素抑制脂肪体中卵黄蛋白的合成,导致血淋巴中卵黄蛋白含量的下降,从而抑制了卵母细胞的发育。抑卵激素并不抑制卵母细胞对卵黄原蛋白的摄取。卵发育神经激素可以颉抗抑卵激素的抑制作用。抑卵激素无种属特异性。     

A specific subunit of vitellogenin that mediates receptor binding

Vitellogenin, an estrogen-induced serum protein synthesized in the liver, is composed of two Mr 250K polypeptides. It is specifically transported by a receptor-mediated endocytic process into the developing oocytes of virtually all oviparious animals. Following endocytosis, in the chicken, vitellogenin is specifically processed to yield several smaller products including the phosvitins (PV) and the lipovitellins (LV). These products are then stored within the oocyte until they are degraded during embryogenesis to provide nutrients for the developing embryo. Direct binding studies using iodinated vitellogenin demonstrate that vitellogenin binds to isolated oocyte membranes with a KD of 2.5 microM. Competition studies indicate that PV is a competitive inhibitor of vitellogenin binding. This leads us to propose that the PV portion of the circulating vitellogenin molecule mediates binding and uptake. Direct binding studies using iodinated PV show that PV binds to isolated oocyte membranes with a KD of 2.4 microM. Competition studies also demonstrate that 3.1 microM vitellogenin inhibits 50% of control 125I-PV binding, but IgG and bovine serum albumin at concentrations up to 10 microM have no effect on 125I-PV binding. Another series of competition experiments using a constant amount of vitellogenin and increasing amounts of 125I-PV indicate that vitellogenin acts as a competitive inhibitor of PV binding and has a KI of 2-3 microM. These results support our hypothesis that the receptor which mediates vitellogenin binding and uptake recognizes determinants on the PV portion of the native vitellogenin molecule.     

Inhibition of (3H)vitellogenin uptake by isolated amphibian oocytes injected with cytoplasm from progesterone-treated mature oocytes.

Fully grown meiotically immature (germinal vesicle stage) amphibian oocytes incorporate radioactive protein ([³H]vitellogenin) following in vitro culture. In vitro exposure of such oocytes to exogenous progesterone induces germinal vesicle breakdown and inhibits incorporation of vitellogenin. In the present studies, we have investigated the effects of cytoplasm taken from mature and immature oocytes on incorporation of vitellogenin and nuclear breakdown following microinjection of this material into immature oocytes. Vitellogenin incorporation was markedly suppressed in oocytes which underwent nuclear breakdown following injection with cytoplasm from mature oocytes. Incorporation of vitellogenin into oocytes which did not mature after injection with cytoplasm taken from mature oocytes resembled that seen in oocytes injected with immature cytoplasm. The degree of suppression of vitellogenin incorporation following cytoplasmic injections was similar to that seen in uninjected oocytes treated with progesterone. Oocytes injected with cytoplasm obtained from immature oocytes did not undergo either nuclear breakdown or changes in vitellogenin incorporation. The results suggest that cytoplasm obtained from mature oocytes contains a factor(s) which alters directly or indirectly the capacity of the oocyte cell membrane to incorporate vitellogenin. Enucleated immature oocytes also incorporated [³H]vitellogenin, and injection of such oocytes with mature, but not immature, oocyte cytoplasm suppressed vitellogenin incorporation. Suppressive effects of injected cytoplasm thus appear to be mediated through physiological changes in the recipient oocyte cytoplasm rather than the nuclear component.     

Vitellogenesis in Xenopus laevis and chicken: cognate ligands and oocyte receptors. The binding site for vitellogenin is located on lipovitellin I

Vitellogenesis is the process of yolk formation in rapidly growing oocytes of oviparous species. The transport of yolk precursor proteins from the blood plasma into the oocyte is achieved by receptor-mediated endocytosis. Although the Xenopus oocyte is one of the prime experimental systems for expression of foreign genes and their products, the receptor for the main vitellogenic protein, vitellogenin, from this extensively utilized cell has not been identified. Here we have applied ligand and immunoblotting to visualize the Xenopus laevis oocyte receptor for vitellogenin as a protein with an apparent Mr of 115,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. The receptor from the amphibian oocyte also recognizes chicken vitellogenin, and vice versa; furthermore, the two receptor proteins are immunologically related as revealed by Western blotting with anti-chicken vitellogenin receptor antibodies. The receptors from both species bind the lipovitellin moiety of vitellogenin, as revealed by ligand blotting with radiolabeled lipovitellin polypeptides as well as by a novel reverse ligand blotting procedure utilizing nitrocellulose-immobilized ligand. Since vitellogenins of chicken and Xenopus have been shown to be structurally similar and evolutionarily related (Nardelli, D., van het Schip, F. D., Gerber-Huber, S., Haefliger, J.-A., Gruber, M., AB, G., and Wahli, W. (1987) J. Biol. Chem. 262, 15377-15383), it appears that conservation of key structural elements required for efficient vitellogenesis extends from the ligands to their receptors on the oocyte plasma membrane.     

Intracellular transport of vitellogenin in Xenopus oocytes: An autoradiographic study

The role of primordial yolk platelets (PYPs) in the transport of the yolk precursor vitellogenin to the yolk platelets in Xenopus laevis oocytes has been demonstrated by electron microscopic autoradiography. Within 20 min after exposure of the oocyte to ³H-labeled-vitellogenin, silver grains are associated with small PYPs which are formed by the fusion of endosomes. At 40 min after incorporation of ³H-labeled vitellogenin, autoradiographic silver grains are associated with larger PYPs and with the superficial layer of yolk platelets. Thus, the results demonstrate that PYPs are an intermediate in the transport of vitellogenin from endosomes to yolk platelets. These observations are consonant with the general hypothesis that vitellogenin first associates (binds?) with the plasma membrane, then is incorporated by endocytosis into endosomes which fuse to form PYPs, and finally the contents of the PYPs are eventually deposited into yolk platelets.     

Inhibition of yolk formation in locust oocytes by trypan blue and suramin

Summary Trypan blue and suramin inhibit receptor-mediated endocytosis of vitellogenin in Locusta migratoria. Both drugs bind to cationic side chains of the vitellogenin molecule, which are presumably also the binding sites for the specific vitellogenin receptor. Thus binding of vitellogenin to the receptor is prevented in isolated oocytes and in oocyte membrane preparations. Small amounts of trypan blue may unspecifically enter oocytes by piggy-back endocytosis. Suramin has a high affinity for vitellogenin and in contrast to trypan blue it does not form insoluble complexes with the protein. Therefore, it may be a useful tool for further analysis of the locust vitellogenin receptor.     

Oosorption in the stink bug,Plautia crossota stali: induction and vitellogenin dynamics

Oosorption, resorption of developing oocytes in the ovary, in P. c. stali is characterized by changes in appearance of oocytes from opaque greyish green or orange to transparent, degeneration of yolk granules and disappearance of oocyte contents. Starvation and virginity were indicated to be factors that induce oosorption. SDS PAGE/Western blotting analysis using anti-vitellogenin antiserum detected two major and many minor bands in haemolymph samples. Egg extracts showed a more complicated set of positive bands in the same analysis. Yolk protein, vitellin, therefore, seemed to be formed after complicated processing of vitellogenin following its uptake by the oocytes. In starved, oosorption-induced females, vitellogenin concentration in the haemolymph was lower than that of fed females, and Western blotting failed to detect either oosorption-specific or ovary-specific peptide fragments in haemolymph samples collected from those females. These results suggest that once oosorption was induced vitellogenin/vitellin in oocytes was degraded rapidly and released into the haemolymph in the form of amino acids or small peptides too small to be recognized by the anti-vitellogenin antiserum.     

Identification and characterization of the vitellogenin receptor in Macrobrachium rosenbergii and its expression during vitellogenesis

In oviparous organisms, oocyte maturation depends on massive production of the egg yolk-precursor protein, vitellogenin (Vg). Vg is taken up by the developing oocytes through receptor-mediated endocytosis (RME), a process essential to successful reproduction. The aims of this study were to identify and characterize the yet-unknown vitellogenin receptor (VgR) from the pleocyamate crustacean Macrobrachium rosenbergii, and to investigate its expression levels during vitellogenesis and its interaction with Vg. The VgR gene was cloned, and its translated protein was specifically located at the oocyte membrane. Moreover, for the first time, a VgR protein was identified and sequenced by mass spectrometry. The putative MrVgR displayed high sequence similarity to VgRs from crustaceans, insects, and vertebrates, and its structure includes typical elements, such as an extracellular, lipoprotein-binding domain (LBD), EGF-like, and O-glycosylation domains, a transmembrane domain, and a short, C-terminal, cytosolic tail. In this article, we identify the first crustacean VgR protein, and present data demonstrating its high affinity for a Vg column followed by elution with suramin and EDTA. Additionally we demonstrate that VgR expression in the oocyte is elevated during vitellogenesis. Our results contribute to the fundamental understanding of oocyte maturation in crustaceans, and particularly elucidate Vg uptake through RME via the VgR.     

STEROID INHIBITION OF PROTEIN INCORPORATION BY ISOLATED AMPHIBIAN OOCYTES

The relationship between blood protein (vitellogenin) incorporation and nuclear maturation was studied in individual amphibian oocytes after in vitro exposure to desoxycorticosterone acetate (DOCA). Isolated Rana pipiens oocytes were incubated in vitro with radioactively labeled oocyte yolk precursor ([³H]vitellogenin) obtained from estrogenized Xenopus laevis. Incorporation of labeled vitellogenin into the oocytes continued over a 24-h period. Oocytes simultaneously exposed to DOCA and to labeled vitellogenin exhibited both inhibition of vitellogenin incorporation and stimulation of nuclear maturation and cortical changes. Inhibition of vitellogenin incorporation was observed after approximately 9 h of incubation and was correlated with the time of nuclear breakdown. Preincubation of oocytes in steroid for 9 h essentially terminated vitellogenin incorporation. Incorporation of vitellogenin occurred after removal of follicle cells from the oocyte by a short treatment with EDTA. These results demonstrate the macromolecular vitellogenin transport system remains operative in oocytes which can undergo nuclear maturation and that the steroid DOCA can affect its function. Evidence suggests that the mechanism of steroid inhibition is in part the result of inhibition of the micropinocytotic process in the oocyte cortex.