Molecular Characterization of Prothoracicotropic Hormone (PTTH) from the Giant SilkmothAntheraea pernyi:Developmental Appearance of PTTH-Expressing Cells and Relationship to Circadian Clock Cells in Central Brain

Harvard University, Cambridge, Massachusetts, United States
Developmental Biology (Impact Factor: 3.55). 10/1996; 178(2):418-29. DOI: 10.1006/dbio.1996.0228
Source: PubMed


Using a PCR strategy, we have cloned the cDNA for prothoracicotropic hormone (PTTH) from the giant silkmoth, Antheraea pernyi. The A. pernyi PTTH cDNA encodes a preprohormone of 221 amino acids that is 51 and 71% identical at the amino acid level with Bombyx mori and Samia cynthia ricini PTTHs, respectively. Bacterially expressed, recombinant A. pernyi PTTH stimulates adult development when injected into debrained pupae. PTTH protein (ca. 30 kDa by Western blot) and mRNA (ca. 0.9 kb by Northern blot) are expressed in brain. Immunocytochemistry and in situ hybridization show that PTTH protein and mRNA are colocalized in L-NSC III from Day 4 of embryogenesis through adult life, with little variation in either protein or mRNA levels at the various ecdyses. A pair of cells expressing immunoreactivity for the circadian clock protein PER is located in the same region as PTTH-expressing L-NSC III in A. pernyi brain. However, double-label immunocytochemical studies show that PTTH and PER are located in different cells. The close anatomical location between PTTH- and PER-expressing cells suggests routes of communication between these two cell populations that may be important for the circadian control of PTTH release.

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    • "PTTH was first purified from the silkworm B. mori as a 30-kDa peptide, consisting of two identical subunits linked with a disulfide bond (Kawakami et al., 1990; Kataoka et al., 1991). Until now PTTH has been identified in Samia cynthia ricini (Ishizaki and Suzuki, 1994), Antheraea pernyi (Sauman and Reppert, 1996), Hylophora cecropia (Sehnal et al., 2002), Manduca sexta (Shionoya et al., 2003), and Helicoverpa zea (Xu et al., 2003). In Lymantria dispar caterpillars this neurohormone was identified by Kelly et al. (1991) and its molecular mass was determined as 11–15 kDa. "
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    ABSTRACT: Lymantria dispar, as most invasive insect species, is very adaptable and reacts quickly to changing environment. Neuroendocrine system first reacts to stress in insects, and specific neurohormonal reorganization may be used in early heavy metal risk assessment. Prothoracicotropic neurohormones (PTTH) control ecdysteroid synthesis (morphogenetic and stress hormones) in insects. In this article, we report the presence of PTTH immunoreactive molecules in L2' dorsolateral neurosecretory neurons (nsn) in caterpillar brains and changes after exposure to pollutant stress of different intensity. For 3 days, after molting into the 4th instar, caterpillars of Lymantria dispar were fed with a high wheat germ diet without (control) or with added cadmium (experimental groups: 10, 30, 100, 250 μg Cd/g dry food weight). Changes in PTTH producing L2' nsn and differences in the intensity of protein bands in the region of PTTH molecular mass (Mr 11-15 kDa) were analyzed. The number of L2' neurons tended to decrease except in the group given the highest cadmium concentration (250 μg). The neurons were enlarged after acute treatment especially in the group given the highest cadmium concentration. The size of L2' nsn nuclei was decreased only in the group fed with 30 μg Cd. Protein band intensity in the Mr region of PTTH remained unchanged in all groups except for the group given the diet with the highest Cd concentration. © 2012 Wiley Periodicals, Inc. Environ Toxicol, 2012.
    Environmental Toxicology 05/2014; 29(7). DOI:10.1002/tox.21804 · 3.20 Impact Factor
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    • "During this course of study, we noticed not only MT-ir but also serotonin receptors (5HTRs)-ir in PTTH-ir cells. The neurosecretory cells (ns cells) secreting PTTH were located in the dorsolateral protocerebrum (DL) of A. pernyi [6], and this condition was also found in Bombyx mori and Manduca sexta [12,13]. cDNAs encoding PTTH from B. mori, M. sexta and A. pernyi were successfully cloned and sequenced [14,15,6]. "
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    ABSTRACT: The release of prothoracicotropic hormone, PTTH, or its blockade is the major endocrine switch regulating the developmental channel either to metamorphosis or to pupal diapause in the Chinese silk moth, Antheraea pernyi. We have cloned cDNAs encoding two types of serotonin receptors (5HTRA and B). 5HTRA-, and 5HTRB-like immunohistochemical reactivities (-ir) were colocalized with PTTH-ir in two pairs of neurosecretory cells at the dorsolateral region of the protocerebrum (DL). Therefore, the causal involvement of these receptors was suspected in PTTH release/synthesis. The level of mRNA(5HTRB) responded to 10 cycles of long-day activation, falling to 40% of the original level before activation, while that of 5HTRA was not affected by long-day activation. Under LD 16:8 and 12:12, the injection of dsRNA(5HTRB) resulted in early diapause termination, whereas that of dsRNA(5HTRA) did not affect the rate of diapause termination. The injection of dsRNA(5HTRB) induced PTTH accumulation, indicating that 5HTRB binding suppresses PTTH synthesis also. This conclusion was supported pharmacologically; the injection of luzindole, a melatonin receptor antagonist, plus 5th inhibited photoperiodic activation under LD 16:8, while that of 5,7-DHT, induced emergence in a dose dependent fashion under LD 12:12. The results suggest that 5HTRB may lock the PTTH release/synthesis, maintaining diapause. This could also work as diapause induction mechanism.
    PLoS ONE 11/2013; 8(11):e79381. DOI:10.1371/journal.pone.0079381 · 3.23 Impact Factor
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    • "Seven cysteine residues are present within the PTTH monomer at conserved locations, as in other known PTTHs [20]. In situ hybridization revealed that MabPTTH is expressed in two dorsolateral neurosecretory cells in each brain hemisphere (Fig. 1C), as are the PTTH genes of other lepidopteran insects [15], [22], [27]. "
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    ABSTRACT: Diapause is a programmed developmental arrest that has evolved in a wide variety of organisms and allows them survive unfavorable seasons. This developmental state is particularly common in insects. Based on circumstantial evidence, pupal diapause has been hypothesized to result from a cessation of prothoracicotropic hormone (PTTH) secretion from the brain. Here, we provide direct evidence for this classical hypothesis by determining both the PTTH titer in the hemolymph and the PTTH content in the brain of diapause pupae in the cabbage army moth Mamestra brassicae. For this purpose, we cloned the PTTH gene, produced PTTH-specific antibodies, and developed a highly sensitive immunoassay for PTTH. While the hemolymph PTTH titer in non-diapause pupae was maintained at high levels after pupation, the titer in diapause pupae dropped to an undetectable level. In contrast, the PTTH content of the post-pupation brain was higher in diapause animals than in non-diapause animals. These results clearly demonstrate that diapause pupae have sufficient PTTH in their brain, but they do not release it into the hemolymph. Injecting PTTH into diapause pupae immediately after pupation induced adult development, showing that a lack of PTTH is a necessary and sufficient condition for inducing pupal diapause. Most interestingly, in diapause-destined larvae, lower hemolymph titers of PTTH and reduced PTTH gene expression were observed for 4 and 2 days, respectively, prior to pupation. This discovery demonstrates that the diapause program is already manifested in the PTTH neurons as early as the mid final instar stage.
    PLoS ONE 04/2013; 8(4):e60824. DOI:10.1371/journal.pone.0060824 · 3.23 Impact Factor
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