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.

Expression of the crustacean hyperglycaemic hormones and the gonad-inhibiting hormone during the reproductive cycle of the female American lobster Homarus americanus.

Crustacean reproduction is regulated by a complex chain of hormonal interactions in which the crustacean hyperglycaemic hormones A and B (CHH-A and CHH-B) and the gonad-inhibiting hormone (GIH) play a primary role. These neurohormones are produced in the same neuroendocrine cells of the X-organ sinus gland complex, situated in the eyestalks of the American lobster, Homarus americanus. In order to obtain more information on the synthesis, storage, release and function of these three neuropeptides during the reproductive cycle, we studied the levels of their mRNAs in the X-organ, their peptide storage in the sinus gland and their concentration in the haemolymph at different stages of the female reproductive cycle. A high CHH-A mRNA level was found only in the previtellogenic stage, while elevated mRNA levels were determined for CHH-B in the mature as well as the previtellogenic stage. High CHH storage levels in the sinus gland were found during previtellogenesis. The total amount of CHH (CHH-A plus -B) in the haemolymph was significantly higher during maturation. A low level of GIH mRNA in the X-organ and a low amount of the GIH I isoform in the sinus gland were found only in the immature stage. In contrast, GIH haemolymph levels were high during the immature and previtellogenic stages. We conclude that CHH-A and -B are involved in triggering the onset of vitellogenesis and that CHH-B in particular is responsible for stimulating oocyte maturation before spawning, while GIH prevents the start of vitellogenesis in the ovary. Moreover, our results show that the balance between the haemolymph levels of the CHHs and GIH may tune the synchronization of reproduction and molting during the biannual reproductive cycle of the American lobster.

Cloning and expression of two mRNAs encoding structurally different crustacean hyperglycemic hormone precursors in the lobster Homarus americanus.

The crustacean hyperglycemic hormone (CHH) of the X-organ sinus gland complex is a multifunctional neurohormone primarily involved in the regulation of blood sugar levels. HPLC analysis of lobster sinus glands revealed two CHH-immunoreactive groups, each consisting of two isoforms with identical amino acid sequences and molecular weights. In order to obtain more information concerning the number and sequences of preproCHHs, and to study their expression, we isolated two full-length cDNAs encoding two different CHH preprohormones. Both preprohormone structures consist of a signal peptide, a CHH-precursor-related peptide and a highly-conserved CHH peptide. Expression studies revealed that the X-organ is not the only source of CHH mRNA because the ventral nerve system also expresses this mRNA. Based on these findings and earlier studies on the effect of eyestalk ablation, implantation of thoracic/abdominal ganglia as well as the multifunctionality of CHH, we postulate that CHH, present in the ventral nerve system is a good candidate for a supplementary role in the control of reproduction and molting.

Cloning and expression of mRNA encoding prepro-gonad-inhibiting hormone (GIH) in the lobster Homarus americanus.

The gonad-inhibiting hormone (GIH) is produced in the eyestalk X-organ sinus gland complex of male and female lobsters, and plays a prominent role in the regulation of reproduction, e.g. inhibition of vitellogenesis in female animals. To study this neurohormone at the mRNA level, we cloned and sequenced a cDNA which encodes GIH in the lobster Homarus americanus. The structure of preproGIH consists of a signal peptide and the GIH peptide itself. A comparative analysis revealed that lobster GIH, together with crab molt-inhibiting hormone, belongs to a separate group of the crustacean hyperglycemic hormone (CHH) peptide family which seems to be unique for crustaceans. Expression studies showed that GIH mRNA is expressed in the eyestalk, indicating that the neuroendocrine center in this optic structure is the only source of GIH. As this center modulates the other (neuro)endocrine organs in crustaceans, it is postulated that GIH regulates production and release of hormones involved in reproduction/molting processes.

Evidence for a conformational polymorphism of invertebrate neurohormones. D-amino acid residue in crustacean hyperglycemic peptides.

Several large peptidic neurohormones have been isolated in crustaceans. In lobster and other related species, each of these neurohormones, and particularly the crustacean hyperglycemic hormone, occurs as two isoforms having the same peptidic sequence and molecular mass. We report here that these isoforms differ by the configuration of a single amino acid residue. The third residue (Phe3) of the lobster hyperglycemic hormones is in either the L- or D-configuration. In addition, we have shown that the biological activity of the two isoforms differs when considering the kinetics of their hyperglycemic effect.

Involvement of eyestalk factors in the neuroendocrine control of osmoregulation in adult American lobster Homarus americanus.

Osmoregulatory capacity (OC) decreased by approximately 50% after eyestalk ablation in adult Homarus americanus when in dilute media. OC was used to assay sinus gland extracts. Injection of total extracts and of some HPLC-separated fractions of sinus glands into destalked lobsters increased OC. One of the described crustacean hyperglycemic hormone isoforms influences osmoregulation. Another fraction of the sinus gland extracts modifies osmoregulation but its nature remains unknown. Variations in OC were examined in response to ecdysterone, Phe-Met-Arg-Phe-NH2, and atrial natriuretic factor but effects were minimal.

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)

Cloning and sequence analysis of cDNA encoding two crustacean hyperglycemic hormones from the lobster Homarus americanus.

Using the polymerase chain reaction with degenerated oligonucleotides, we have isolated cDNA clones that encode two structurally different (92% identity) crustacean hyperglycemic hormones (CHH) from the lobster Homarus americanus. The deduced amino acid sequences fully agree with previously published data on partial amino acid sequences, amino acid compositions and molecular masses of hyperglycemic peptides in the lobster. A comparative analysis between the deduced primary structure of two lobster CHH and the crab CHH sequence reveals a phylogenetic relationship and allows the prediction of biologically important regions within the structures of these novel neuropeptides.

Comparative characterization of hyperglycemic neuropeptides from the lobster Homarus americanus.

With the use of a two-step HPLC purification procedure, two sets of two isoforms of the crustacean hyperglycemic hormone (CHH) were isolated from sinus glands of the lobster Homarus americanus. Structural differences between the two groups of isoforms were found in their amino acid sequences, amino acid compositions and precise molecular weights. Using peptide mapping, the difference between the isoforms in each group was located within the first eight amino acids at the N-termini. The nature of this difference remained unclear as all four peptides had the same N-terminal amino acid sequence unto residue 19.