Demonstration of a cell-specific isomerization of invertebrate neuropeptides.

Neurohemal organs of the lobster Homarus americanus contain isoforms of the crustacean hyperglycemic hormone, which differ by the third amino acid (phenylalanyl) residue that is either in the L- or in the D-configuration. Polyclonal antisera have been raised in rabbit against synthetic octapeptides with the sequence corresponding to the N-terminal part of the L- or D-phenylalanine-containing isoforms. Their specificity was shown by immunoassays, indicating that they discriminate the isoforms of the lobster hyperglycemic neuropeptides. It was demonstrated that the two major forms of the crayfish Orconectes limosus hyperglycemic hormone also correspond to peptide isomers containing the L- or D-phenylalanyl residue. The cellular distribution of the isoforms among the neurosecreting cells of the major neuroendocrine complex in lobster and crayfish has been studied by immunohistochemistry. Every hyperglycemic hormone-containing cell was labelled with the anti-L antisera while only some of them were visualized with the anti-D antisera. These results constitute the first observation of peptide isomerization at the cellular level and suggest that the isomerization process occurs in specialized neuroendocrine cells.

Localization of a crustacean hyperglycemic hormone-like immunoreactivity in the neuroendocrine system of Euscorpius carpathicus (L.) (Scorpionida, Chactidae).

The neuroendocrine system of Euscorpius carpathicus was immunohistochemically localized using a polyclonal antiserum raised against a purified Homarus americanus crustacean hyperglycemic hormone (Hoa-cHHA). There were cross-reactions in E. carpathicus procerebral and subesophageal neurosecretory cells, neurohemal organs, and intra- and extraganglionic neurosecretory tracts. Among the neurohemal structures, the Kwartirnikov's organ, the Tropfenkomplex, and the coxal disc reacted strongly. In Euscorpius, the differing results between adults and juveniles suggest neurosecretory variations related to developmental stage. These immunohistochemical observations suggest the presence of substances related to the cHH in scorpions; however, in this heterologous system, it is not at present possible to assess physiological significance.

Neuropeptides and related nucleic acid sequences detected in peneid shrimps by immunohistochemistry and molecular hybridizations.

The neurosecretory cells in the eyestalks of Penaeus indicus and P. vannamei were studied by immunocytochemistry using polyclonal antisera raised against purified Homarus americanus neuropeptides. Cross-reactions between two H. americanus anti-crustacean hyperglycemic hormone antisera and Penaeus neurosecretory material were observed. The specific anti-vitellogenesis inhibiting hormone antiserum only showed an immunological reaction in the nervous tract and the sinus gland of Penaeus, suggesting a progressive exposure of a characteristic epitope which was amenable to immunological detection. Molecular hybridizations were performed in P. indicus and P. vannamei with a digoxigenin tailed 23mer oligonucleotide probe deduced from two partial sequences of uncharacterized, purified P. duorarum neuropeptides. Two distinct clusters of positive cells were observed by in situ hybridization experiments in the medulla terminalis ganglionic X-organ. Classical control tests gave negative results. Northern and Southern blot analyses were performed with the same tailed probed and allowed the determination of molecular weights for the mRNA and for a DNA restriction fragment (Pst 1), 1.7 kb and 200 bp, respectively. These observations show the existence of a strong homology between the P. duorarum sequence (selected for synthesizing the probe), and some P. indicus and P. vannamei neuropeptide sequence(s). Heterologous antisera were tested in other Arthropods to complete our analyses. In the centipede Lithobius forficatus and the scorpion Euscorpius carpathicus, the anti-crustacean hyperglycemic hormone antiserum induced a strong cross-reaction. A monoclonal anti-bombyxin-1 antiserum showed an immunoreaction in the neurosecretory system of the insects Tenebrio molitor and Labidura riparia. In contrast, the anti-bombyxin-1 antiserum did not react either in Penaeus or in Lithobius, and the Homarus hyperglycemic hormone antiserum did not react in the insects that were tested. A comprehensive view of these observations is discussed in relation to a divergence in Arthropod evolution.

Localization of messenger RNAs encoding crustacean hyperglycemic hormone and gonad inhibiting hormone in the X-organ sinus gland complex of the lobster Homarus americanus.

The localization of messenger RNAs encoding the crustacean hyperglycemic hormone, involved in regulation of carbohydrate metabolism and the gonad inhibiting hormone, which inhibits vitellogenesis, was studied in the eyestalk of the lobster Homarus americanus using complementary RNA probes for in situ hybridization. For the detection of gonad inhibiting hormone messenger RNA, we cloned and sequenced a partial complementary DNA encoding lobster gonad inhibiting hormone and for crustacean hyperglycemic hormone messenger RNA detection an available complementary DNA was used. This approach reveals that there is a frequent but inconsistent cellular co-localization of the two neurohormones. Furthermore, our data show that male lobsters contain an equal number of neuroendocrine gonad inhibiting hormone cells as female lobsters. An additional study, involving the use of in situ hybridization in combination with immunocytochemistry, shows that the synthetic activity of the crustacean hyperglycemic hormone- and gonad inhibiting hormone-producing cells can be followed at the messenger RNA as well as the protein level. This reveals that when strong immunostaining is present, the messenger RNA staining is usually weak or absent and vice versa. In conclusion, the presence of cells, containing only gonad inhibiting hormone messenger RNA or only crustacean hyperglycemic hormone messenger RNA, indicates that lobster crustacean hyperglycemic hormone and gonad inhibiting hormone originate from two different precursors. Co-localization of the two neurohormone messenger RNAs confirms the co-localization at the peptidergic level found by immunocytochemistry and thus these findings were not due to cross-reactions between the two antisera.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of the mRNA encoding vitellogenesis inhibiting hormone in neurosecretory cells of the X-organ in Homarus americanus by in situ hybridization.

Vitellogenesis inhibiting hormone (VIH)-mRNA in secretory cells of the eyestalk of Homarus americanus was detected by nonradioactive in situ hybridization (ISH) using two digoxigenin-tailed oligonucleotide probes deduced from the peptide sequence. Two distinct clusters of positive cells were observed in the medulla terminalis ganglionic X-organ (MGTX). Only one of them gave a strong immunoreaction after incubation with a specific polyclonal anti-VIH serum and corresponded to the conventionally described VIH producing cells. The significance of the cells reacting positively in ISH but not in immunocytochemistry (ICC) is discussed. Northern blot analysis using 32P-labeling confirms the specificity of the probes and indicates an approximate size of 2.5 kb for VIH mRNA.