James T. Bradley

Monoclonal antibody-based immunoassay of vitellogenin in the blood of male channel catfish (Ictalurus punctatus).

1. A monoclonal antibody to vitellogenin of channel catfish (Ictalurus punctatus) was made, and its specificity was demonstrated using Western blots of serum from female fish, estradiol-treated male fish, untreated male fish, vitellogenin purified by three different methods and egg extracts. 2. An enzyme-linked immunosorbent assay (ELISA), using this monoclonal antibody, detected vitellogenin in the plasma of 59 out of 60 untreated 17-24-month-old male channel catfish with a mean concentration of 338 micrograms/ml and a maximum concentration of 4240 micrograms/ml. 3. Vitellogenin levels in male channel catfish were unrelated to testicular stage, gonadosomatic index and month.

Vitellogenin induction by estradiol in channel catfish, Ictalurus punctatus

In oviparous vertebrates estrogens induce hepatic synthesis of vitellogenin (VG), a blood protein sequestered in vitellogenic oocytes and from which lipovitellin (LV) and phosvitin are derived. Our objective was to identify VG in the channel catfish, Ictalurus punctatus. An intraperitoneal injection of estradiol-17 beta into adult male fish induced a dose-dependent accumulation of a 150 kDa protein (EP) in the plasma. EP was detectable in Coomassie blue-stained polyacrylamide gels within 24 hr after injection of 2 mg hormone/100 g body weight. During the next 4 days, EP increased from 5 to about 25% of the total plasma protein. Electrophoretic mobility, peptide mapping, and immunological crossreactivity showed EP to be indistinguishable from a plasma protein in adult females with vitellogenic ovaries. Two major yolk polypeptides, YP1 (120 kDa) and YP2 (29.6 kDa), were precipitated by (NH4)2SO4 from a yolk protein extract. YP1 but not YP2 reacted with an anti-EP polyclonal antiserum in Western blots. Peptide mapping after proteolysis with trypsin showed YPs 1 and 2 to be unique and revealed structural homologies between YP1 and EP. Liver but not pancreatic explants from an estradiol-treated male synthesized and secreted a [35S]methionine-labeled, 150 kDa protein beginning about 2 hr after initial exposure to the label. We tentatively conclude that EP and YP1 represent VG and LV, respectively. YP2 remains unidentified.

Effects of triazoles on fungi. III. Composition of a plasma membrane-enriched fraction of Taphrina deformans *

Comparative chemical analyses were conducted with plasma membrane-enriched fractions of Taphrina deformans cells grown in a medium with or without the C-14 demethylation inhibitor propiconazole at a concentration that gives 50% growth inhibition. The membrane fractions were prepared using differential and discontinuous sucrose density gradient centrifugation, and characterized by cytochemical, enzymatic and chemical analyses. Membranes of nontreated cells were similar to those from other fungi with a protein/lipid ratio of 1.2, 13% phospholipid content in the membrane lipid (122 μg/mg protein), and a relatively high sterol/phospholipid molar ratio of 0.69. The corresponding membrane fraction from propiconazole-treated cells had 24% less lipid, 27% less phospholipid, 5-times more triacylglycerol relative to other neutral acyl lipids, and over a 2-fold higher sterol/phospholipid ratio. The greater sterol/phospholipid ratio was due to a higher C-14 methyl sterol content rather than less functional sterol (brassicasterol). Membranes from treated cells contained slightly less protein than those from nontreated cells, but there was little difference in the electrophoretic separation patterns of solubilized membrane polypeptides.

Seasonal levels of serum vitellogenin and oocyte growth in the channel catfish Ictalurus punctatus

Abstract Seasonal levels of serum vitellogenin (VG) were measured in adult and juvenile (10–30 months old) female channel catfish using an enzyme-linked immunosorbent assay (ELISA). In adults sampled during a 2-year period, the highest mean (±s.e.) VG level was 30.21 ± 5.36 mg/ml in May before spawning and declined to 3.79 ± 0.89 mg/ml in June after the fish had spawned. Oocyte diameter increased from September to November, remained the same until February, then increased again until maximum mean diameter of 2356 ± 49 μm was measured in May. Vitellogenin was detected in the serum of juvenile fish that were 12 months old, but yolk granules were not visible in hematoxylin and eosin stained sections of oocytes until the fish were 18 months old. Mean serum VG levels of juvenile fish were lower than the adults, and no VG peak was observed in the juvenile fish. Vitellogenin was present in the serum throughout the year in adult fish and in juveniles older than 12 months.

Vitellin processing and protein synthesis during cricket embryogenesis

At the start of insect embryogenesis most of the protein mass of the egg cytoplasm exists as vitellin (Vt) obtained endocytically during vitellogenesis. Of the new embryo polypeptides (EP) appearing in the egg during embryogenesis, many are synthesized de novo, while, in some species, others derive from developmentally programmed partial proteolysis of Vt. Earlier we showed that by the end of vitellogenesis the two native Vts in Acheta domesticus exist in opposing gradients along the longitudinal axis of the egg. Here we hypothesize that this ooplasmic Vt distribution presents a milieu for Vt processing out of which region-specific regulatory molecules could arise. The metabolic origin and stage-specific patterns of seven predominant EPs (EP 1-7) identified by SDS-PAGE were examined and the results correlated with developmental morphology during the 14 days of embryogenesis. Based on antibody reactivity, peptide mapping and in vitro radiolabeling, we determined that EPs 1-3, 6 and 7 are Vt-derived, while EPs 4 and 5 are produced de novo by the embryo. The five Vt-derived EPs appear during the first 24 h of embryogenesis when migrating cleavage nuclei and associated cytoplasm form the cellular blastoderm, and levels of EPs 4 and 5 increase during days 4-6 of embryogenesis when katatrepsis and yolk mass contraction occur. Positive periodic acid-Schiff staining indicated that EPs 1-3 and their Vt-precursor polypeptides are glycoproteins. This work shows that developmental stage-specific Vt processing occurs during A. domesticus embryogenesis and points next to investigation of the functional significance of Vt cleavage products during development.

Early detection and juvenile hormone-dependence of cricket vitellogenin

Abstract The timing of in vivo induction of vitellogenin synthesis in the house cricket, Acheta domesticus (L.), was examined using an immunodiffusion assay with an estimated sensitivity of 6.3 μg vitellogenin/ml haemolymph. The time course of vitellogenin appearance in adult female haemolymph and analysis of abdominal autonomy for vitellogenin production in females decapitated at various times after the moult revealed that induction does not normally occur during the first 12 h of adult development but is completed by 14–16 h in some females. This is much earlier than expected on the basis of electrophoretic analyses previously reported but correlates with the early rise in juvenile hormone titre recently observed by other workers. Decapitation/hormone replacement experiments using juvenile hormones I and III confirmed the juvenile hormone control of cricket vitellogenin production. Newly moulted adult females were markedly refractory to the inductive effects of juvenile hormone; however, among adult females decapitated at the moult but aged 24 h before injection, 74% produced vitellogenin in response to juvenile hormone. These studies provide the temporal framework for future analyses of juvenile hormone-dependent fat body changes which precede initiation of vitellogenin synthesis.

Vitellogenin uptake and vitellin localization in insect follicles examined using monoclonal antibodies and confocal scanning microscopy

Summary Confocal scanning immunofluorescent microscopy and monoclonal antibodies were used to examine the route of uptake of vitellogenin (VG) by vitellogenic follicles and the ooplasmic localization of vitellin (VN) in the cricket, Acheta domesticus, and the stick insect, Carausius morosus. Uptake and cytoplasmic regionalization of a non-vitellogenic sulfated protein, sp 157/85, by C. morosus oocytes were also examined. By indirect immunofluorescence VG in both species and sp 157/85 were visualized in spaces between follicle cells and in peripheral yolk spheres. One cricket VG polypeptide had a regionalized distribution in the folliclular epithelium, and VN polypeptides in both species and sp 157/85 in C. morosus had regionalized distributions within the ooplasm. Localization of sp 157/85 to the anterior pole of the oocyte appeared to be stage-specific.

Vitellogenesis in the allatectomized stick insect Carausius morosus (br.) (Phasmatodea: Lonchodinae)☆

Abstract Effects of allatectomy on vitellogenesis in adult Carausius morosus Br. were examined using rocket immunoelectrophoresis, polyacrylamide gel electrophoresis, fluorographic identification and quantitation by liquid scintillation of in vivo35S-methionine-labeled proteins, and light microscope autoradiography. In normal adults and in adults allatectomized as last instar nymphs, Coomassie Blue-stained vitellogenin (Vg) was first detectable in the hemolymph between 3 and 5 days after adult emergence. Allatectomy of 11–18-day-old adults produced no detectable effects on subsequent Vg synthesis, secretion or uptake, but ovarian follicles in adults that had been allatectomized as nymphs were less efficient at taking up Vg than were those in sham-operated animals. Compared to sham-operated controls, adults allatectomized as nymphs displayed an increased rate of accumulation of newly synthesized Vg in the hemolymph, a decreased rate of accumulation of vitellin in terminal follicles, a decrease in the size of yolk spheres containing newly synthesized vitellogenic protein in the peripheral ooplasm, and alterations in the size distribution of terminal follicles. Thus, post-induction vitellogenesis and primary induction of Vg synthesis in C. morosus do not require active corpora allata (CA), but a normal rate of Vg uptake may be JH-facilitated.

Ultrastructure of the corpus cardiacum and corpus allatum of the house cricket Acheta domesticus

SummaryThe ultrastructure of the corpus cardiacum (CC) and corpus allatum (CA) of the house cricket, Acheta domesticus, is described. Axon profiles within the CC contain neurosecretory granules 160–350 nm in diameter which are indistinguishable from those found in type I neurosecretory cells of the pars intercerebralis and in the nervus corporis cardiaci I. The CC itself contains two cell types: intrinsic neurosecretory cells and glial cells. Intrinsic NSC cytoplasm contains Golgi bodies and electron dense neurosecretory granules 160–350 nm in diameter. Synaptoid configurations with 20–50 nm diameter electron lucent vesicles were observed within axon profiles of the CC. The structure of the CA is relatively uniform with one cell type predominating. Typical CA cells possess large nucleoli, active Golgi complexes, numerous mitochondria, and occassional microtubules. Groups of dark staining cells scattered throughout the CA of some animals were interpreted as evidence of cellular death.

Vitellogenin synthesis and secretion in ovariectomized crickets

Abstract 1. 1. Rabbit antiserum against egg vitellin was used to measure in vivo vitellogenin (Vg) concentrations, synthesis and secretion in adult Acheta domesticus ovariectomized prior to adult ecdysis. 2. 2. Hemolymph and fat body Vg concentrations were higher in 10-day-old ovariectomized adults than in sham-operated animals of the same age. 3. 3. Ovariectomy resulted in an increased rate of incorporation of 14 C-leucine into fat body Vg. 4. 4. Ovariectomy resulted in a selective decrease in the rate of secretion of newly synthesized Vg. 5. 5. The insect fat body may provide a model for regulation of protein secretion in eukaryotic cells.