A fast method to study the secretory activity of neuroendocrine cells at the ultrastructural level.

Cryo field emission scanning electron microscopy (cryo-FE-SEM) is a versatile technique that allows the investigation of the three-dimensional organization of cells at the ultrastructural level over a wide range of magnifications. Unfortunately, cryopreparation of the specimens for this technique remains cumbersome, in particular because ice crystal formation must be prevented during freezing. Here we report that a light prefixation with glutaraldehyde and incubation in glycerol as cryoprotectant or a high-pressure freezing approach are both excellent procedures for cryopreparation of animal cells to be used in combination with cryo-FE-SEM. Using the proopiomelanocortin-producing intermediate pituitary melanotrope cells of Xenopus laevis as a physiologically inducible neuroendocrine system, we compared the ultrastructural characteristics of inactive and hyperactive neuroendocrine cells. The overall quality of the ultrastructural images was comparable for the two cryopreparation procedures, although some fine structures were better conserved using high-pressure freezing. Melanotrope cells in a secretory inactive state contained numerous storage granules and a poorly developed endoplasmic reticulum (ER), while large amounts of rough ER were present in hyperactive cells. Thus, the cryo-FE-SEM approach described here allows a fast ultrastructural study on the secretory activity of neuroendocrine cells.

Automated nanoflow liquid chromatography-tandem mass spectrometry for a differential display proteomic study on Xenopus laevis neuroendocrine cells.

Many proteomic projects based on a comparison of protein profiles displayed on two-dimensional polyacrylamide gel electrophoresis rely on the identification of these proteins using peptide mass fingerprinting on a matrix-assisted laser desorption/ionization mass spectrometer after tryptic digestion. However, this approach is limited to an organism of which genomic information is largely available, i.e. when the total genome sequence is known. For other organisms, mass spectrometric sequence analysis is necessary for protein identification. We established a nano-LC-MS-MS system based on a quadrupole time-of-flight mass spectrometer, which allows automated sequence analysis of tryptic digestion mixtures from single gel spots. This system is applied in a differential-display proteomic study to identify differentially expressed proteins in the neuroendocrine cells of the neurointermediate pituitary of black- and white-background adapted Xenopus laevis.

Localization of p24 putative cargo receptors in the early secretory pathway depends on the biosynthetic activity of the cell.

Members of the p24 family of putative cargo receptors (subdivided into p24-alpha, -beta, -gamma and -delta) are localized in the intermediate-and cis-Golgi compartments of the early secretory pathway, and are thought to play an important role in protein transport. In the present study, we wondered what effect increased biosynthetic cell activity with resulting high levels of protein transport would have on the subcellular localization of p24. We examined p24 localization in Xenopus intermediate pituitary melanotrope cells, which in black- and white-adapted animals are biosynthetically highly active and virtually inactive respectively. In addition, p24 localization was studied in Xenopus anterior pituitary cells whose activity is not changed during background adaptation. Using organelle fractionation, we found that in the inactive melanotropes and moderately active anterior pituitary cells of white-adapted animals, the p24-alpha, -beta, -gamma and -delta proteins are all located in the Golgi compartment. In the highly active melanotropes, but not in the anterior cells of black-adapted animals, the steady-state distribution of all four p24 members changed towards the intermediate compartment and subdomains of the endoplasmic reticulum (ER), most probably the ER exit sites. In the active melanotropes, the major cargo protein pro-opiomelanocortin was mostly localized to ER subdomains and partially co-localized with the p24 proteins. Furthermore, in the active cells, in vitro blocking of protein biosynthesis by cycloheximide or dispersion of the Golgi complex by brefeldin A led to a redistribution of the p24 proteins, indicating their involvement in ER-to-Golgi protein transport and extensive cycling in the early secretory pathway. We conclude that the subcellular localization of p24 proteins is dynamic and depends on the biosynthetic activity of the cell.

Daily variations in crustacean hyperglycaemic hormone and serotonin immunoreactivity during the development of crayfish.

The present study investigated changes in crustacean hyperglycaemic hormone (CHH) and serotonin (5-hydroxytryptamine, 5-HT) immunoreactivity in the retina and the X-organ/sinus gland complex (XO-SG) of the crayfish Procambarus clarkii at two developmental stages, post-embryonic stage two (PO2) and the juvenile stage, at three different times of day, under a photoperiod cycle of 12 h:12 h L:D, using qualitative and quantitative immunohistochemical methods. In the retina, CHH immunoreactivity is located in the tapetal cells, while 5-HT immunoreactivity is found in the retinular cells. In the XO-SG, CHH-immunoreactivity is localized to the CHH-producing cell perikarya and in their axons and endings in the sinus gland, while 5-HT immunoreactivity is restricted to axon endings branching into the perikarya of the CHH-producing cells. A stereological analysis demonstrates that the PO2 and juvenile stages show significant differences in the amount of the immunoreactive CHH and 5-HT material at the three selected time points, indicating daily and related changes in the levels of CHH and 5-HT in the XO-SG and the retina. Our findings therefore support the idea that daily rhythms in the secretory activity of the XO-SG complex affect the circadian sensitivity of the eye. Furthermore, the differences found between the PO2 and juvenile stages suggest that both CHH and 5-HT are key factors in the development of the circadian rhythm of retinal sensitivity.

Involvement of crustacean hyperglycemic hormone in the control of gill ion transport in the crab Pachygrapsus marmoratus.

Total extracts of sinus glands (SG) of the euryhaline grapsid crab Pachygrapsus marmoratus contain peptidic factor(s) that stimulate osmoregulatory processes in isolated and perfused posterior gills from crabs acclimated to dilute seawater. This study investigated the nature of the active factor(s). Separation of P. marmoratus SG peptides by reverse-phase HPLC, followed by a direct enzyme-linked immunosorbent assay using an anti-Carcinus maenas crustacean hyperglycemic hormone (CHH) antiserum, identified a major immunoreactive chromatographic peak. A glucose quantification bioassay demonstrated a strong and specific hyperglycemic activity following injection of the immunoreactive peak, therefore defined as the CHH of P. marmoratus. Isolated posterior gills were then perfused with HPLC fractions using a dose of 4 SG equivalents/assay. The CHH fraction consistently and significantly increased the transepithelial potential difference and Na(+) influx by about 50%. The effect was rapid and reversible. Another substance of unknown nature (eluted earlier than CHH in the HPLC gradient) caused a small increase in Na(+) influx (14%) but had no effect on the transepithelial potential difference. No other peptidic product from the SG had significant effect on the measured osmoregulatory parameters. These results indicate that CHH, in addition to its hyperglycemic activity, is also implicated in the control of branchial ionic transport. This neuropeptide may thus constitute a major factor involved in the control of osmoregulation in decapod crustaceans.

Induction of proopiomelanocortin mRNA expression in animal caps of Xenopus laevis embryos.

To convert animal pole cells of a frog embryo from an ectodermal fate into a neural one, inductive signals are necessary. The alkalizing agent NH4Cl induces the expression of several anterior brain markers and the early pituitary marker XANF-2 in Xenopus animal caps. Here it is demonstrated that NH4Cl also induced proopiomelanocortin (POMC)-expressing cells (the first fully differentiated pituitary cell type) in stage 9 and 10 Xenopus animal caps, and that all-trans retinoic acid, a posteriorizing agent, was able to block this induction when it was administered within 2 h after the start of NH4Cl incubation. Thus, after 2 h, the fate of Xenopus animal cap cells was determined. Microinjection of ribonucleic acid (RNA) encoding noggin, an endogenous neural inducer, led to the induction of POMC gene expression in animal caps of stage 10 embryos, suggesting that noggin represents a candidate mesodermal signal leading to the POMC messenger (m) RNA producing cell type in uncommitted ectoderm. Hence, an alkalizing agent and a neural inducer can generate a fully differentiated POMC cell lineage from Xenopus animal caps.

Differential onset of expression of mRNAs encoding proopiomelanocortin, prohormone convertases 1 and 2, and granin family members during Xenopus laevis development.

The production of peptide hormones through proteolytic cleavage of prohormones, e.g., proopiomelanocortin (POMC), involves a number of regulated secretory proteins, such as prohormone convertase PC1, PC2 and granin family members, that are co-expressed with the prohormone. Although the expression of these proteins has been well-studied in adult animals, data on their expression during development are limited. We used whole-mount in situ hybridization to visualize POMC mRNA expression in the intermediate and anterior pituitary of Xenopus tadpoles. A more sensitive analysis, namely semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) on total RNA isolated from Xenopus developmental stages, revealed that the expression of POMC, PC1 and PC2 mRNA commenced at stages 13 (neural plate stage), 15 (neural fold stage) and 19 (neural tube stage), respectively, with a gradual increase in their expression levels during further development. Surprisingly, and in contrast to what holds for POMC and the convertases, mRNAs for secretogranin II and III (SgII, SgIII) and 7B2 were not only expressed during neural development, but could already be detected in unfertilized mature oocytes, the first cleavage stages and in blastula-stage embryos. These granins are thus maternally present in Xenopus embryos suggesting that they may have a role during oogenesis and/or early embryonic development.

Dynamics of proopiomelanocortin and prohormone convertase 2 gene expression in Xenopus melanotrope cells during long-term background adaptation.

The toad Xenopus laevis is able to adapt its skin color to background light intensity. In this neuroendocrine reflex, the proopiomelanocortin (POMC)-derived peptide alpha-melanophore-stimulating hormone (alphaMSH) is a key regulatory factor. In animals adapting to a black background, release of alphaMSH from the pituitary pars intermedia causes dispersal of melanin in skin melanophores. To investigate the long-term in vivo dynamics of alphaMSH production during black background adaptation, the biosynthetic rate of POMC and the contents of POMC, alphaMSH and the POMC processing enzyme precursor convertase 2 (PC2) have been studied in the pars intermedia using pulse-labeling, Western blot and radioimmunoassay. In control animals, adapted to a white background, the rate of POMC biosynthesis and the POMC content were low, while high alphaMSH and PC2 contents were found. After 1 week of adaptation to a black background, the rate of POMC biosynthesis and the POMC protein content had increased 19- and 3.7-fold respectively. These parameters attained a maximum level (28- and 5. 8-fold higher than control) after 3 weeks and remained at these elevated levels for at least 12 weeks. After 1 week, the pars intermedia content of alphaMSH was only 30% of the control level, but after 6 and 12 weeks, the alphaMSH level had increased to the control level. The PC2 content decreased to 52% of control after 1 week and stabilized after 3 weeks at a level slightly lower than the control value. The results show that during long-term background adaptation a steady-state situation is reached, with a balance between the biosynthesis, enzymatic processing and release of alphaMSH. The in vivo dynamics of the processing enzyme PC2 suggest a parallel storage and release of alphaMSH and mature PC2 in the Xenopus pituitary pars intermedia.

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.

Molecular biology of neurohormone precursors in the eyestalk of Crustacea.

Our knowledge concerning the primary structures of crustacean neuropeptides has been broadened considerably during the last few years and has greatly contributed to the successful application of molecular biological techniques to crustacean neuroendocrine research. In this review, we compare and discuss the preprohormones of the Red Pigment Concentrating Hormone (RPCH), the Pigment-Dispersing Hormone (PDH) and the different members of the Crustacean Hyperglycemic Hormone, Molt-Inhibiting and Gonad-Inhibiting Hormone family (CHH/MIH/GIH peptide family), recently elucidated by cloning and sequencing of the respective cDNAs. Expression studies, using in situ hybridization, Northern blots and RNase protection assays, have demonstrated that the mRNAs encoding some of the aforementioned preprohormones (for example, preproPDH and preproCHH) are not only expressed in the eyestalk but also in other parts of the central nervous system. The combination of molecular biological techniques with (bio)chemical and immunochemical methods provides elegant tools to study neuropeptides at the level of mRNA and peptide in individual animals during different physiological conditions. The fundamental knowledge obtained by such a combined approach will give detailed insight into how neuropeptides are involved in the adaptation of Crustacea to a broad spectrum of natural and aquacultural conditions.

Stimulation of osmoregulating processes in the perfused gill of the crab Pachygrapsus marmoratus (Crustacea, Decapoda) by a sinus gland peptide.

Isolated posterior gills of the hyper-hyporegulating crab Pachygrapsus marmoratus were perfused with extracts of homologous sinus glands. Sinus gland extracts stimulated the influx of Na+ ions and increased the transepithelial potential difference in the gills in a dose-dependent and reversible fashion. The bioactivity of extracts prepared from crabs that had been acclimated to 10/1000 salinity for at least 1 week was not significantly different from that of extracts prepared from seawater (36/1000 salinity) crabs. The perfusion experiments with both extracts containing two sinus glands significantly increased Na+ influx by about 150% and transepithelial potential difference by about 45%. Sinus gland extracts also increased the Na+/K(+)-ATPase activity by 54% in incubated posterior gills. The bioactivity of extracts was reduced by pronase and trypsin, but not by heating for 10 min at 100 degrees. The molecular weight of the responsible factor(s) was > 5000 Da. Thus, the sinus gland of P. marmoratus is concluded to be involved in the neuroendocrine control of osmoregulation and to contain a peptide(s) that directly influences brachial function.

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.

Cloning and expression of two crustacean hyperglycemic-hormone mRNAs in the eyestalk of the crayfish Orconectes limosus.

Crustacean hyperglycemic hormone (CHH) is a multifunctional neurohormone produced in the eyestalk of crustaceans and is primarily involved in the regulation of carbohydrate metabolism. In several crustacean species, CHH isoforms with identical amino acid sequences and molecular masses, but with different chromatographic elution patterns, are synthesized. To obtain sequence information on the CHH preprohormone in the crayfish Orconectes limosus we isolated two full-length cDNAs encoding two structurally different preproCHH species. The sequences of these precursors differ slightly in the signal peptide, the CHH-precursor-related peptide(CPRP)-coding sequences and in the non-coding regions, but are identical in the CHH peptide-coding sequence. Determination of the levels of preproCHH mRNAs and the amount of CHH peptide in the eyestalks of individual animals revealed that the ratio between the two preproCHH mRNAs varies for different individuals while the ratio between the two CHH peptide isoforms does not differ among animals. Our results suggest that the existence of two CHH isoforms in the crayfish O. limosus is due to a post-translational modification event. Northern-blot analysis showed only one band in eyestalk tissue with a size of approximately 2.4 kb, similar to the sizes of the cDNA sequences. Southern-blot analysis revealed the presence of at least two preproCHH genes in the crayfish suggesting a gene duplication event. Slight modifications in the duplicated genes could be responsible for the existence of the two preproCHH-encoding mRNAs.

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.

Structure and localization of mRNA encoding a pigment dispersing hormone (PDH) in the eyestalk of the crayfish Orconectes limosus.

The pigment-dispersing hormone (PDH) is produced in the eyestalks of Crustacea where it induces light-adapting movements of pigment in the compound eye and regulates the pigment dispersion in the chromatophores. To study this hormone at the mRNA level, we cloned and sequenced cDNA encoding PDH in the crayfish Orconectes limosus. The structure of the PDH preprohormone consists of a signal peptide, a PDH precursor-related peptide (PPRP) and the highly conserved PDH peptide at the carboxy-terminal end. In situ hybridization in combination with immunocytochemistry revealed four cell clusters expressing PDH in the optic ganglia of the eyestalk. Three clusters stained both with the PDH cRNA probe and the PDH antiserum, however, the perikarya in the lamina ganglionaris (LG) only stained with the PDH antiserum, suggesting the presence of a PDH-like peptide in the LG.

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.