John K. Koeppe

On the metabolism of N-acetyldopamine in Periplaneta americana.

N-acetyldopamine is rapidly and extensively converted to N-acetyldopamine 3-O-phosphate and N-acetyldopamine 3-O-sulphate in the newly ecdysed cockroach, Periplaneta americana. Dopamine 3-O-sulphate also serves as a naturally occurring precursor of N-acetyldopamine 3-O-sulphate in vivo. Radioisotope experiments revealed that the N-acetyldopamine moiety of these esters is incorporated into the cuticle during sclerotization and that the phosphate and sulphate moieties are not (not, at least, as the intact esters). It is suggested that N-acetyldopamine 3-O-phosphate and N-acetyldopamine 3-O-sulphate may be the forms in which the eventual cuticular sclerotizing agent (N-acetyldopamine?) is transported from the blood into the epidermis by a ‘carrier’ protein.

Juvenile hormone regulation of structural changes and DNA synthesis in the follicular epithelium of Leucophaea maderae

Abstract Juvenile hormone (JH I) stimulates specific morphological and biochemical changes in the follicular epithelium surrounding the terminal oocytes in Leucophaea maderae . These include extracellular and intracellular structural changes, increased rates of follicle cell DNA synthesis, and elevated follicle cell DNA concentrations. Using females decapitated 24 hr after ecdysis, we have shown that JH I injections stimulate the following structural changes in the follicular epithelium: the appearance of channels between adjacent follicle cells and of spaces between the follicular epithelium and the maturing oocyte; an increase in follicle cell size; the development of an extensive rough endoplasmic reticulum system; and an enlarged nucleus within each follicle cell. No increase in the number of follicle cells surrounding the developing terminal follicles is found in 7-day JH I-treated females, although the terminal follicles are almost twice as long as those in untreated females. In addition, we have demonstrated that JH stimulates the following biochemical events in the ovary: a 3.5 fold increase in thymidine incorporation into follicle cell DNA, with no subsequent transfer of such DNA to the developing oocyte, and a 1.4 fold increase in ovarian DNA in 7-day JH-treated females. These data indicated that JH stimulates follicle cell DNA synthesis. The absence of any corresponding division of follicle cells suggests that JH I may induce polyploidy in follicle cells. Extended exposure of decapitated females to JH I does not result in complete ovarian maturation. Although fat bodies in the treated insects continue to display an increasing rate of vitellogenin synthesis, DNA synthesis in the terminal follicles declines rapidly after day 9, and the terminal follicles ultimately degenerate.

Assay and identification of juvenile hormone binding proteins in Leucophaea maderae.

We modified a binding assay using polyethylene glycol (PEG) to precipitate bound hormone. Optimum precipitation occurred when reaction mixtures were incubated with 10-40% PEG and 1.25-2.5 mg/ml gamma-globulins for 2-90 min at 4 or 23 degrees C. Results from this assay and from the dextran-coated charcoal assay were similar. Addition of phenylmethylsulfonyl fluoride eliminated nonspecific esterase activity in extracts. JH III-binding macromolecules were identified in hemolymph and ovaries of Leucophaea maderae. These molecules were pronase- and heat-sensitive and saturable. Using Scatchard analysis an average KD of 2.04 (+/- 0.32) X 10(-8) M and 1.91 (+/- 0.80) X 10(-8) M was calculated for hemolymph and ovarian binding proteins. JH III had the highest affinity for binding sites, followed by JH I and JH 0. Various extraction procedures caused changes in JH affinity for both binding proteins. At high concentrations the (+) isomer and mixed isomer preparations of methoprene and hydroprene competed for binding sites. Binding proteins had no affinity for the (-) isomer or for the JH III acid.

Structure, haemolymph titre, and regulatory effects of juvenile homone during ovarian maturation in Leucophaea maderae

Abstract Juvenile hormone (JH) synthesized and secreted in vitro by the corpora allata of mated adult Leucophaea maderae females was determined to be JH III (methyl-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate). The haemolymph titre of JH was determined during maturation of the terminal oocytes in the first reproductive cycle of L. maderae. In virgin females, JH is not detectable in the haemolymph during the first eight days following adult emergence; however, by 10 days after emergence, trace quantities of JH are apparent. Mating stimuli induce a dramatic increase in the concentration of haemolymph JH, with a peak occurring approximately 12 days after mating; thereafter, the JH concentration declines until it has reached an undetectable level 19 days after mating, at the time of chorion deposition. During ovarian maturation, changes in the rates of synthesis of vitellogenin by the fat body and DNA by the ovary correlate closely with the haemolymph titre of JH. However, no such correlation exists between the JH titre and the extensive ovarian protein synthesis that occurs in L. maderae coincident with chorion formation. The effects of JH I and JH III on both vitellogenin synthesis and ovarain DNA synthesis are statistically similar.

Relationship of hemolymph juvenile hormone-binding protein to lipophorin in Leucophaea maderae

Abstract Monoclonal antibodies (MAb) specific for the hemolymph 220 kD juvenile hormone-binding protein (JHBP) of the cockroach, Leucophaea maderae, were used to further characterize the JHBP. Spleen cells, from mice immunized with semipurified hemolymph JHBP, were fused with myeloma cells to produce hybridomas secreting antibodies specific for the hemolymph 220 kD JHBP. Positive clones were recloned and rescreened by the same methods, resulting in the establishment of five cell lines producing IgG2a anti-hemolymph JHBP antibodies. Using MAb against the 220 kD hemolymph JHBP, it was demonstrated by Western blot analysis that the 220 kD JHBPs present in both ovarian and egg case extracts have antigenic determinants similar to those of the hemolymph JHBP. In addition, both the 275 and 220 kD JHBPs, synthesized and secreted by fat body maintained in vitro, were detected by anti-hemolymph JHBP antibody, demonstrating that the two media proteins and the JHBP in the ovary share identical antigenic determinants. To determine if there is a relationship between hemolymph JHBP and lipophorin, hemolymph proteins were separated on potassium bromide gradients. Gradient analysis confirmed that the lipophorin complex bound JH and that the 220 kD JHBP is apolipophorin I (apolp I). The apolp I specific MAb did not cross-react with apoliphorin II (apolp II).

Ovarian maturation in Leucophaea maderae: Juvenile hormone regulation of thymidine uptake into follicle cell DNA

Abstract Our research demonstrates that juvenile hormone (JH I) stimulates thymidine incorporation into ovarian follicle cell DNA in the ovoviviparous cockroach, Leucophaea maderae. A rapid, quantitative method for monitoring 3H-thymidine incorporation into ovarian DNA, in vitro, is described. Cultured ovarian tissue from L. maderae incorporates 3H-thymidine into DNA at a linear rate between 16 and 120 min; analysis of the incorporated label revealed at least 98% of it to be in DNA. Using L. maderae females that had been mated 7 days after adult emergence, we monitored the following biochemical phenomena during the 18–22 day period of terminal oocyte growth: (1) 3H-thymidine incorporation into ovarian DNA: (2) general protein synthesis in fat body; and (3) specific fat body vitellogenin synthesis. Decapitation of mated females with maturing oocytes arrested both ovarian DNA synthesis and fat body vitellogenin synthesis. Substantial restoration of both types of synthesis was induced by injection of JH I. The resumption of thymidine incorporation into DNA was localized in the follicular epithelium of the terminal oocyte. In decapitated virgin females, injection of JH I stimulated oocyte growth and 3H-thymidine incorporation into ovarian DNA. Dose and time response curves indicate that peak stimulation of ovarian DNA synthesis occurred between 72 and 96 hr after administration of a single optimal dose of 25 μg JH I. The concurrent manifestation of 3H-thymidine uptake into ovarian DNA and activity within the fat body indicates that a similar hormonal mode of action may be operative with respect to both tissue types in virgin females.

Photoaffinity labelling of juvenile hormone binding proteins in the cockroach Leucophaea maderae

Abstract The synthesis and testing of several diazocarbonyl JH analogs (diazo JHA) which act as photoaffinity labels for insect juvenile hormone binding proteins are described. The best competitor, 10,11-epoxyfarnesyl diazoacetate, has been shown to irreversibly reduce [3H]-JH III binding to both ovarian and hemolymph JHBP from Leucophaea maderae after irradiation at 254 nm for 20 seconds. No loss of activity was observed after incubation of JHBP and diazo JHA without irradiation. Protection from photoinactivation by diazo JHA II was achieved by the presence of an equimolar amount of JH III during the photolysis. Photoaffinity labeled proteins show loss of binding capacity without alteration of the binding affinity. This is the first example of the use of a photoaffinity label in the study of JH action on a molecular level, and may become a valuable tool in the elucidation of JH-receptor-chromatin interactions.

Synthesis and secretion of a precursor hemolymph juvenile hormone-binding protein in the adult female cockroach Leucophaea maderae

Abstract Hemolymph juvenile hormone-binding protein (JHBP) is synthesized and secreted from fat body in the adult female cockroach, Leucophaea maderae . The data in this paper suggest it is initially secreted from the fat body as a larger peptide whereas data in the accompanying paper demonstrate that JHBP is apolipophorin I. Using media from cultures of fat body maintained in vitro , a JH-binding component was found that is JH III saturable, has a K D of 1.5 × 10 −8 M, binds JH III > JH II > JH I, and has a sedimentation value of 6.5S on high salt sucrose gradients. Each of these properties is identical to those of the JHBP extracted from the hemolymph. To identify the protein that bound JH, media proteins were photoaffinity labeled with 10-[10,11- 3 H]epoxyfarnesyl diazoacetate ([ 3 H]EFDA). The results revealed that two media proteins bound [ 3 H]EFDA in the absence of JH III, but not in the presence of 100-fold excess JH III. The molecular weights of the two media peptides were estimated by SDS-PAGE to be 275,000 and 220,000. To determine if the JHBP found in media of fat body cultures was due to hemolymph contamination of fat body, incorporation of [ 3 H]leucine into newly synthesized and secreted fat body proteins during a 48 h culture period was monitored. During the culture period, linear increases in the concentrations of radiolabeled 275 and 220 kD JHBP were observed. Monoclonal antibodies specific for the 220 kD hemolymph JHBP were found to recognize both the 275 and 220 kD JHBPs in the media. To investigate the possibility that the 275 kD protein is a precursor to the 220 kD protein and that components of the hemolymph process or modify the precursor, hemolymph was introduced into fat body cultures and relative concentrations of the 275 and 220 kD media JHBPs were determined. Addition of hemolymph to these organ cultures resulted in an increase in the concentration of radiolabeled 220 kD JHBP and a proportional decrease in the concentration of radiolabeled 275 kD JHBP, suggesting that the 275 kD protein is a precursor to the 220 kD hemolymph JHBP. The mechanism of processing or modification remains undetermined.

Changes in follicle cell morphology, ovarian protein synthesis and ovarian DNA synthesis during oöcyte maturation in Leucophaea maderae: Role of juvenile hormone

Abstract Changes in follicle cell morphology were correlated with changes in rates of protein synthesis and DNA synthesis by the ovary during ovarian maturation in Leucophaea maderae. During the vitellogenic period of oocyte development, which lasts approx, 15 days, morphological changes in the follicle cells are accompanied by moderate rates of ovarian protein synthesis and rapid rates of ovarian DNA synthesis. At approx. 15 days after mating, the shape of the follicle cells changes from cuboidal to squamous, ovarian DNA synthesis is arrested, and ovarian protein synthesis increases slightly. During the final period of oocyte development, which lasts approx, two days, the interfollicular channels between the follicle cells have disappeared and the squamous follicle cells, which contain an extensive rough endoplasmic reticulum, deposit a chorion around the mature oocyte. These morphological changes are accompanied by a radical increase in ovarian protein synthesis, while ovarian DNA synthesis remains arrested. Immediately before ovulation, ovarian protein synthesis starts to decline, reaching a minimal level 24 hr post-ovulation. Ovarian maturation is dependent on the presence of juvenile hormone (JH) only during the vitellogenic stage of oocyte development. Decapitation of insects at any point during the first 10 days after mating arrests the synthesis of DNA and retards the synthesis of protein by the ovary, resulting in degeneration of the oocyte. Subsequent injection of JH restores both events to normal levels within 72 hr. Decapitation on or after the tenth day following mating does not alter normal oocyte development, chorion deposition, ovulation or egg case formation. Primary induction of protein synthesis in ovaries from virgin females can be achieved by either an in vivo or in vitro exposure of the tissue to JH, thus confirming a site of action for JH to be ovarian tissue. Electrophoretic analysis of the soluble proteins from JH-exposed ovaries in vivo reveals that JH stimulates general protein synthesis, rather than the synthesis of a specific major protein such as vitellogenin.

Follicle cell polyploidy in Leucophaea maderae: Regulation by juvenile hormone

Abstract Microspectrophotometric analysis of Feulgen-stained nuclei of the terminal follicle cells in the cockroach Leucophaea maderae showed that during maturation the follicle cells became polyploid. In virgin females, the follicle cell nuclei were diploid. After mating, and during vitellogenesis, the ploidy of the follicle cells increased from 2 C to 32 C with a small percentage of 64 C nuclei. There was no further increase in the ploidy levels during the chorionic stage of development. Injections of juvenile hormone III into decapitated virgin females elevated the ploidy levels in the follicle cells. The DNA content of these nuclei at 96–120 h after injection of juvenile hormone III increased from 2 C to 4 C. Such polyploidization of nuclei was dose-dependent with the highest DNA content occurring in response to 25–50 μg juvenile hormone III. The juvenile hormone-induced increase in DNA content correlated with an increase in the rate of [ 3 H]thymidine incorporation into DNA. Our data suggest that the role of juvenile hormone in follicle cell development during the vitellogenic period, whether direct or indirect, is to promote selectively a large increase in the DNA content of the cells. This may facilitate the next stage of follicle cell development, choriogenesis.