Temporal events of gypsy moth vitellogenesis and ovarian development

Abstract The vitellogenic period of gypsy moth ovarian development starts on day 3 of the pupal stage and continues through adulthood. During this period, rapid increases occur in follicle size, protein content, and wet weight of the ovary. Patency is observed on day 3 of the pupal stage.
Pre-vitellogenic follicles are formed in the last larval stadium. Newly formed follicles detach from the germarium on day 4, and increase rapidly to 140 per ovariole at the end of the last larval stadium. The pre-vitellogenic follicles are uniformly around 50 um in diameter. No vitellogenin is incorporated into the oocytes until the pupal stage.
Polyacrylamide gel electrophosesis (PAGE) in the presence of sodium dodecylsulphate (SDS) analysis of male and female haemolymph samples and vitellogenic ovaries demonstrates the presence of two female-specific subunits of vitellogenin of 180 kD and 160 kD. These proteins are detected only in haemolymph and ovarian extracts of vitellogenic females. The molecular weight of the native protein determined by size exclusion chromatography is approximately 400–420 kD.
A highly sensitive double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed to monitor the temporal changes in vitellogenin titre in haemolymph. Vitellogenin production starts on day 2 of the last larval stadium, reaching a maximum level by day 6 of the last larval stadium, and decreasing in the late pupal stage as vitellogenin was internalized into the oocytes. This is the first report of vitellogenin production occurring in the larval stage of a holometabolous insect. The fact that vitellogenin production and uptake occur during different stages of development in the gypsy moth, opens up some interesting questions concerning the underlying regulatory mechanisms controlling each process.     

Yolk sphere formation is initiated in oocytes before development of patency in follicles of the moth,Plodia interpunctella

We describe a provitellogenic stage, a previously unrecognized stage of follicle development in moths, and show that oocytes begin yolk sphere formation prior to the development of patency by the follicular epithelium. The vitellogenic activities of follicles from pharate adult femalePlodia interpunctella (Hübner) were determined by visualizing the subunits of vitellin (YP1 and YP3) and the follicular epithelium yolk protein (YP2 and YP4) using monospecific antisera to each subunit to immunolabel whole-mounted ovaries or ultrathin sections. At 92 h after pupation, yolk spheres that contained only YP2 began to proliferate in the oocytes. The inter-follicular epithelial cell spaces were closed at 92 h making vitellogenin inaccessible to the oocyte, and consequently, the vitellin subunits were not observed in the yolk spheres. YP2 uptake most likely occurred across the brush border from the follicular epithelial cells to the oocyte at this time. At 105 h, the inter-follicular epithelial cell spaces appeared closed yet trace amounts of labeling for vitellin were observed in the spaces and also in the yolk spheres along with YP2. Equivalent labeling for all four YPs in yolk spheres was finally observed at 112 h after pupation when the follicular epithelium had become patent. These data indicate that the provitellogenic stage is an extended transition period between the previtellogenic and vitellogenic stages that lasts for approximately 13 h, and it is marked at the beginning by YP2 yolk sphere formation in the oocyte and at the end by patency in the follicular epithelium.     

Moulting hormone,juvenile hormone and the ultrastructure of the fat body of adult Sarcophaga bullata (Diptera)

Summary In the ovoviviparous fly, Sarcophaga bullata, vitellogenesis is cyclic; a process reflected in ultrastructural changes in the fat body cells and oenocytes. At eclosion the larval fat body has not yet completely disappeared. During vitellogenesis the fat body cells are specialized for intensive protein synthesis showing a very extensive RER and numerous invaginations of the plasma membrane. These features disappear when the eggs descend into the oviducts to complete embryogenesis. The predominant feature of the oenocytes is their very prominent SER. The fat body cells of the males are never as specialized for protein synthesis as those of the females. Feeding of ecdysterone to males for 3 or more days induces a rather extensive subcellular apparatus for protein synthesis, i.e., invaginations of the plasma membrane and an extensive RER. Juvenile hormone is completely ineffective in this respect. Both ecdysterone and juvenile hormone have pronounced but different effects on the oenocytes of males.     

Reproductive patterns in seventeen species of warmwater fishes in a Missouri River reservoir

Synopsis The timing of ovarian maturation and spawning of 17 warmwater fish species in Lake Oahe (South and North Dakota) was estimated from changes in the mean ovary indices (ratios of ovary weight to fish length). The onset of vitellogenesis varied within species (up to 2 months). Maturation of the ova took from 7.5 to 10 months, depending on species. Annual variations in the mean date of peak spawning of individual species during 1964–1971 were usually less than a week. There was little overlap of the annual mean peak spawning dates of the 17 species, and an established sequence of spawning among species was shown. A relatively high incidence of atresia in the shovelnose sturgeon, northern pike, and carp indicated that these species had apparently not yet adapted to the altered and variable spawning conditions in this reservoir. Regularity of spawning would seem to provide the best chance for spawning success in variable environments such as Lake Oahe.     

Identification of the yolk receptor protein in oocytes of Nereis virens (Annelida,Polychaeta) and comparison with the locust vitellogenin receptor

Summary In oviparous animals large amounts of yolk proteins of extraovarian origin are accumulated by developing oocytes during vitellogenesis. The yolk protein precursors, the vitellogenins (VTG), are transported into the oocytes by receptor-mediated endocytosis. In oocytes of the polychaetous annelid, Nereis virens, the receptor protein for VTG was visualized by ligand blotting studies as a protein with an apparent molecular mass of 190 kDa under non-reducing conditions. Anti-Locusta VTG receptor antibodies recognize the Nereis VTG receptor protein. The Nereis VTG receptor protein binds Locusta and Schistocerca VTG; the VTG receptor proteins of both locust species bind the Nereis vitellin. These results indicate the conservation of structural elements important for internalization of VTG.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane-sulphonic acid - HBS HEPES-buffered saline - PAP peroxidase-anti-peroxidase - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - TRIS, TBS TRIS-buffered saline - VT vitellin - VTG vitellogenin     

Studies on the ovotestis of the slug Agriolimax reticulatus (Müller)

Summary The ovarian oocytes of Agriolimax reticulatus (Müller) have been studied by light and electron microscopy and electron cytochemistry. The development of the oocyte in the ovotestis may be divided into three stages.During Stage I the oocyte cytoplasm contains mainly ribosomes and also strands of endoplasmic reticulum, scattered mitochondria and Golgi systems. The nucleus contains both a paranucleolus and an eunucleolus. By Stage II the oocyte has enlarged, especially in a plane parallel to the basement membrane. In addition to the above mentioned organelles, the cytoplasm contains lipid, glycogen and early yolk platelets. During Stage III, the oocyte continues to enlarge, but mainly in a plane perpendicular to the basement membrane. A considerable degree of cytoplasmic differentiation has also taken place. The plasma membrane of the oocyte has become specialized with the appearance of a polysaccharide-rich glycocalyx, microvilli and pinocytotic tubules. Elsewhere, much of the background cytoplasm, containing Golgi-derived, polysaccharide and acid phosphatase-rich multivesiculate bodies, lipid and glycogen, is sequestered by smooth membranes and ultimately fuses with the growing yolk platelets. The nucleus contains an amphinucleolus, characteristic of many gastropods.The findings of this study are discussed in relation to results from other studies on oogenesis.     

Bluegill (Lepomis macrochirus) vitellogenin: purification and enzyme-linked immunosorbent assay for detection of endocrine disruption by papermill effluent

Vitellogenin (VTG) is a highly specific marker of exposure to environmental estrogens and has been used extensively in field and laboratory studies of estrogenic endocrine disruption in fishes. The purpose of this study was to develop and validate a sensitive, competitive, enzyme-linked immunosorbent assay (ELISA) specific for bluegill (Lepomis macrochirus) vitellogenin. Bluegill VTG was purified by anion exchange chromatography on DEAE-agarose. The polypeptide had an apparent mass of 170 kDa and was specifically recognized by the rabbit antiserum raised against bluegill female-specific plasma protein. Plasma samples from vitellogenic females diluted in parallel with the purified VTG standard curve in the ELISA. The detection limit of the assay was 29 ng/ml and the working range extended to 2700 ng/ml. Recovery of purified VTG was 85.8+/-9.5%, intra-assay variation was 6.4% and interassay variation was 12.3%. We used this ELISA to analyze the seasonal cycle of vitellogenesis in female bluegill and to evaluate potential disruption of this process by exposure to bleached kraft mill effluent (BKME). Captive female bluegill stocked in outdoor experimental streams in New Bern, NC had the lowest levels of VTG, estradiol-17beta (E2), and testosterone (T) and the smallest oocyte diameters in January, but these variables increased in March and remained elevated through August, suggesting an extended spawning season. Plasma VTG, E2, T and oocyte diameter were unaffected by exposure to BKME concentrations as high as 30%. Development of the VTG ELISA allowed rapid and convenient analysis of plasma samples to evaluate exposure to potential endocrine disrupting compounds.     

For uptake of yolk precursors,epithelial cell-oocyte gap junctional communication is required by insects representing six different orders

For uptake of vitellogenin protein into nascent yolk spheres, communication through open gap junction channels between the follicle epithelium and oocyte is required by six different insects representing six different orders. It was recently shown in the hemipteran, Oncopeltus fasciatus, that endocytic uptake of yolk protein resulting in the formation of nascent yolk spheres depended upon an intact epithelium communicating with the oocyte through patent gap junctions. Following treatment with octanol, which down-regulated gap junctions below the level of dye coupling, vitellogenin uptake was terminated. Yet, for another hemipteran, Dysdercus intermedius, it has been shown that yolk spheres can form even when all epithelial cells have been stripped from the oocyte. To determine if the mechanism seen in Oncopeltus is present in other insects, we utilized the same techniques to study nascent yolk sphere production in a dipteran, Drosophila melanogaster, a lepidopteran, Actias luna, a hymenopteran, Xylocopa virginica, a coleopteran, Tenebrio molitor and an orthopteran, Acheta domesticus. In each of these, when gap junctions were down-regulated yolk uptake quickly stopped. That six different insects from six different orders all required a gap junctionally transmitted chemical signal of epithelial cell origin suggests that this mechanism is widespread throughout the insects.     

Molecular analysis of nutritional and hormonal regulation of female reproduction in the red flour beetle, Tribolium castaneum

Female reproduction includes maturation of oocytes and the synthesis of yolk proteins (vitellogenin, Vg) in the fat body and their deposition into the oocytes. Our recent studies showed that juvenile hormone (JH) regulates Vg synthesis and 20-hydroxyecdysone (20E) regulates oocyte maturation in the red flour beetle (Tribolium castaneum). Here, we report on the role of nutritional signaling on vitellogenesis and oogenesis. Comparison of gene expression between fed and starved beetles by microarray analysis showed the up-regulation of genes involved in energy homeostasis and down-regulation of genes involved in egg production in the starved beetles. The RNA interference (RNAi) aided knock-down in the expression of genes involved in insulin and TOR signaling pathways showed that both these signaling pathways play key roles in Vg synthesis and oocyte maturation. Starvation of female beetles resulted in a block in Vg synthesis but not in the progression of primary oocyte development to the resting stage. Feeding after starvation induced Vg synthesis and the progression of primary oocytes from the resting stage to the mature stage. However, in the beetles where JH or 20E synthesis or action was blocked by RNAi, both Vg synthesis and oocyte maturation were affected suggesting that both these hormones (JH and 20E) and nutritional signaling and their cross-talk regulate vitellogenesis and oogenesis.     

Precursor structure of egg proteins in the coral Galaxea fascicularis

In the egg of the reef coral Galaxea fascicularis, four proteins (named GfEP-1 to -4) are stored in high abundance. In the present study, a cDNA containing a full-length open reading frame for GfEP-1 was cloned, and the translated protein sequence was compared to the N-terminal sequences of GfEP-2, -3, and -4. GfEP-1 and -2 were shown to be generated by processing of a precursor of 1439 amino acids, and GfEP-3 turned out to be a partial fragment of GfEP-2. The precursor protein contained regions which exhibited similarities to vitellogenins (Vgs) in bilaterian animals (oviparous vertebrates and invertebrates including nematodes, arthropods, and molluscs). This study reports the first cloning and characterization of a full-length cDNA encoding a Vg in a non-bilaterian animal, and argues that the emergence of Vg as a precursor of egg yolk proteins predated the divergence of the cnidarian and bilaterian lineages.