Allatostatin in ovaries, oviducts, and young embryos in the cockroach Diploptera punctata.

The quantity and localization of -Phe-Gly-Leu-amide allatostatins (-F-G-L-amide AST) was determined by ELISA and immunohistochemistry in ovaries and oviducts and in pre-dorsal closure embryos. AST in the cytoplasm of basal oocytes gradually increased from 4 to 35 fmol/ovary pair from the start (day 2) to the completion of vitellogenesis (day 6), then rapidly increased to 121 fmol/ovary pair during choriogenesis. In oviducts, AST-immunoreactivity was found in nerves to the muscle layer and in epithelial cells. AST-immunoreactivity in oviduct epithelial cells increased during vitellogenesis. A marked increase in quantity of AST in oviduct tissue between completion of chorion formation and immediately after ovulation appears to result from AST released from oocytes as they travel down the oviducts because AST content of newly ovulated eggs was 40% lower than late stage chorionated oocytes, and these oocytes released AST when incubated in saline. AST in embryos, localized in yolk cells, decreased as embryos approached dorsal closure. That this material in ovaries and embryos is AST was confirmed by its ability to inhibit JH synthesis in vitro and identification by MALDI-TOF mass spectrometry of a peptide with a mass corresponding to that of a Diploptera punctata AST. These findings indicate likely novel functions for ASTs: facilitation of ovulation and utilization of yolk.

The effect of ovary implants on juvenile hormone production by corpora allata of male Diploptera punctata.

In the cockroach Diploptera punctata, vitellogenic basal oocytes stimulate juvenile hormone production by the corpora allata. Experiments with males were designed to determine whether oocytes must grow vitellogenically in order to stimulate juvenile hormone production. Two ovarioles with vitellogenic basal oocytes were implanted into unoperated and sham-operated males that do not produce vitellogenin, and males with denervated corpora allata, that produce more juvenile hormone, and sometimes more vitellogenin. Males with corpora allata in similar conditions were injected with saline as controls. In males with denervated corpora allata compared to sham-operated and unoperated males, the implanted basal oocytes showed a greater increase in length, protein, and vitellin content. Juvenile hormone synthesis by denervated corpora allata in males with ovariole implants was greater than in controls. In 10 of 50 males with denervated corpora allata in which one or no ovarioles grew, juvenile hormone production was not higher than in controls. This suggests that if sufficient juvenile hormone is not present to produce vitellogenin, or oocytes do not take vitellogenin up, juvenile hormone production is not stimulated. In sham-operated males implanted with ovarioles, no difference was detected in juvenile hormone synthesis compared to controls. However when unoperated males were used a significant increase was detected. This suggests that intact nerves from the brain to the corpora allata restrained juvenile hormone production so that ovarioles could elicit only slight stimulation of the corpora allata, and oocytes continued vitellogenesis but more slowly than in denervated males. Thus the extent of vitellogenesis appears to determine the ability of ovaries to stimulate juvenile hormone production.

Release of neurosecretory granules within the corpus allatum in relation to the regulation of juvenile hormone synthesis in Diploptera punctata.

The release of neurosecretory granules within the corpora allata (CA) of the viviparous cockroach Diploptera punctata has been compared in glands with intact nerves from the brain (Brain-CA) and those detached from the brain. Measurements of juvenile hormone (JH) synthesis in vitro, comparing these two conditions of the CA at several stages of vitellogenesis in adult females, showed lower production of hormone in Brain-CA complexes than in CA alone. Glands treated with tannic acid to trap exocytotic granules before fixation for electron microscopical examination showed, in sample sections, 10 times more exocytotic profiles in the glands with intact nerves to the brain than in the isolated glands. Sections treated with antibody against allatostatin I (Dip 7), a member of the neuropeptide family that inhibits JH synthesis by CA in vitro, showed neurosecretory granules in allatostatin immunoreactive nerves to be 75+/-4% of the granules in the sample of sections of CA. Because the total quantity of allatostatin in CA was found by ELISA not to vary significantly with changes in JH synthesis, it is concluded that the lower rates of JH synthesis by glands with intact nerves to the brain are most likely due to the release of small amounts of allatostatin within the CA.

Allatostatin in hemocytes of the cockroach Diploptera punctata.

Allatostatins are neuropeptides that inhibit the production, by the corpora allata, of a major insect hormone, juvenile hormone. These peptides are produced by cells of the brain and ganglia as well as by midgut endocrine cells. Transport from these sites may contribute to the allatostatin content in the hemolymph (insect blood). Using a monoclonal antibody against Diploptera punctata allatostatin I (A-P-S-G-A-Q-R-L-Y-G-F-G-L-NH2) and in situ hybridization with a digoxigenin-labeled cRNA probe generated from a portion of the allatostatin gene, it is demonstrated that allatostatin is present in and synthesized by granular hemocytes of D. punctata. About 5% of the hemocytes react with anti-allatostatin antibody and a similar number hybridize with a cRNA probe that detects allatostatin-specific mRNA. Electron micrographs showed that allatostatin-immunoreactive material occurs in membrane-bound, uniformly dense granules that frequently fill fusiform-shaped cells. Allatostatin in cell and plasma fractions of hemolymph quantified by enzyme-linked immunosorbent assay and by bioassay for inhibition of juvenile hormone synthesis in vitro indicated that about equal quantities (0.1-0.2 fmol/microl) are present in cell and plasma fractions. The production of allatostatin by hemocytes suggests that allatostatins may function as regulatory peptides in hemolymph activities in addition to their other known functions.

Two new allatostatins from the brains of Diploptera punctata.

Allatostatins VI and VII have been isolated from saline extracts of the brain of the viviparous cockroach Diploptera punctata. Active fractions, obtained by successive reverse-phase high pressure liquid chromatography separations, inhibited juvenile hormone (JH) III production by corpora allata (CA) in vitro. The primary structures, Y-P-Q-E-H-R-F-S-F-G-L-amide (VI) and D-G-R-M-Y-S-F-G-L-amide (VII), clearly identify them as members of a family of D. punctata allatostatins, five of which have been identified previously and shown to have F-G-L-amide at the C terminus. Synthetic allatostatins VI and VII coeluted with the native allatostatins on two successive high pressure liquid chromatography separations. Allatostatins VI and VII have the same capacity to inhibit JH production by CA of 2 day virgin females as does allatostatin I, previously shown to be a potent inhibitor similar in activity to allatostatin V. Allatostatins VI and VII also resemble the other allatostatins in their ability to affect both larval and adult CA, in the reversibility of their effect, and in the abolition of their effect by addition of farnesoic acid (a late precursor of JH) to CA in vitro.

Allatostatin-immunoreactive neurons projecting to the corpora allata of adult Diploptera punctata.

A monoclonal antibody against allatostatin I was used to demonstrate the allatostatin-immunoreactive pathways between the brain and the corpus cardiacum-corpus allatum complex in the adult cockroach Diploptera punctata. The antibody was two to three orders of magnitude more sensitive to allatostatin I than to the other four known members of the allatostatin family. Whole and sectioned brains in which immunoreactivity was localized with horseradish peroxidase-H2O2-diaminobenzidine reaction showed strongly immunoreactive cells in the pars lateralis of the brain with axons leading to and arborizing in the corpus cardiacum and the corpus allatum. Although many neurosecretory cells of the pars intercerebralis project to the corpora allata only, four strongly immunoreactive cells were evident here (two pairs on either side), and these did not project to the corpus cardiacum and corpus allatum but rather terminated within the protocerebrum in areas in which lateral cells also formed arborizations. Immunoreactivity was found in many other cells in the brain, especially in the tritocerebrum.

Allatostatins in the nerves of the antennal pulsatile organ muscle of the cockroach Diploptera punctata.

The presence of allatostatins in the nerves of the antennal pulsatile organ muscle of the cockroach Diploptera punctata was confirmed by immunocytochemistry, bioassay, and HPLC. Immunocytochemical reactivity with monoclonal antibody against allatostatin I showed strong allatostatin immunoreactivity in the antennal heart nerve which innervates this muscle with varicosities along the muscle fibers and in the insertion of the muscle on the pulsatile ampullae. Bioassay of Sep-Pak purified muscle extract demonstrated inhibition of juvenile hormone synthesis by corpora allata in vitro. A dose-response curve showed maximum inhibition of juvenile hormone synthesis was achieved with 10-20 pulsatile organ muscle eq/corpora allata, and 50% inhibition achieved with an estimated 2.6 pulsatile organ muscle eq. Two successive HPLC separations of the Sep-Pak purified extract yielded bioactive fractions corresponding to the elution times of the five known allatostatins.

Response of male corpora allata to ovarian stimulation in the cockroach, Diploptera punctata.

In Diploptera punctata, female corpora allata (CA) show a cycle of juvenile hormone (JH) synthesis in response to oocyte development. Whether the smaller male CA can also respond to the ovary was investigated. One or two pairs of male CA were implanted into allatectomized females during the period corresponding to egg growth. Low rates and no cycle of JH synthesis occurred in CA of sham-operated males or in male CA implanted into males or ovariectomized females. On the contrary, male CA, either one or two pairs, implanted into females with ovaries showed a cycle of increase and decrease in JH synthesis similar in pattern to that of female glands implanted into females. However, the peak of JH synthesis for a single pair of male CA occurred 1 day later than the peak for two pairs of male CA or one pair of female CA. Also the maximum rates of synthesis of one or two pairs of male CA were lower than those of female glands. However, rates of JH synthesis per cell and per unit volume of corpus allatum tissue were higher for one pair of male CA than for one pair of female CA. Thus, the intrinsic difference between male and female CA appears to be their size rather than rate of JH synthesis and ability to respond to ovarian stimuli.

Primary structure of four allatostatins: neuropeptide inhibitors of juvenile hormone synthesis.

Four neuropeptides that inhibit juvenile hormone synthesis by the corpora allata have been isolated from brains of the virgin female cockroach Diploptera punctata. These allatostatins are 8-13 amino acids long, are amidated, and show sequence similarity, including a 3-amino acid sequence at the C-terminal end that is common to all four peptides. The peptide sequences are as follows: allatostatin 1, Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2; allatostatin 2, Gly-Asp-Gly-Arg-Leu-Tyr-Ala-Phe-Gly-Leu-NH2; allatostatin 3, Gly-Gly-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH2; and allatostatin 4, Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2. An in vitro bioassay of the synthesized allatostatins showed greater than 40% inhibition of juvenile hormone synthesis by corpora allata of virgin females with 10(-9) M allatostatin 1, 10(-8) M allatostatins 2 and 4, and 7 X 10(-7) M allatostatin 3. Inhibition by allatostatins 1-4 was reversible. In addition, allatostatin 1 inhibited juvenile hormone synthesis by corpora allata from mated females and last-instar larvae of D. punctata and corpora allata of adult female Periplaneta americana.

Further evidence of a quantitative deficiency of chain-specific globin mRNA in the thalassemia syndromes.

Formamide gel electrophoresis separates the mRNA fraction from reticulocyte polyribosomes of adult humans into two major RNA species with migratory rates identical to those of the alpha- and beta-globin mRNAs of the rabbit. That these two RNAs of human origin are the globin mRNAs is further supported by the deficiency of the presumed beta mRNA in reticulocyte polyribosomes of fetuses and premature infants, whose cells make gamma chains in preference to beta chains. The globin mRNAs of reticulocyte polyribosomes from patients with hematological disorders were estimated by scanning the stained formamide gels. In contrast to individuals with either hemolytic anemia without hemoglobinopathy or sickle cell anemia who had beta mRNA to alpha mRNA ratios of approximately one, a patient with Hb S-beta-thalassemia had a ratio of beta mRNA to alpha mRNA of 0.75 while two subjects with homozygous beta-thalassemia had severe deficiencies of beta mRNA. Conversely, a patient with alpha-thalassemia (Hb H disease) had a ratio of beta mRNA to alpha mRNA on reticulocyte polyribosomes of 6. These data provide further evidence of a quantitative deficiency of chain-specific globin mRNA in patients with the thalassemia syndromes.