Ovary maturing parsin and diuretic hormone are produced by the same neuroendocrine cells in the migratory locust, Locusta migratoria.

In the migratory locust, the CRF-related diuretic hormone that stimulates fluid secretion by the Malpighian tubules, and the ovary maturing parsin, a neurohormone able to stimulate oogenesis, are produced by the same neuroendocrine cells of the pars intercerebralis in the brain.

In vitro peptidergic neurons from the adult locust pars intercerebralis: morphological and immunocytological studies.

Primary cell cultures were prepared from a major neurosecretory center of the adult locust brain, the pars intercerebralis, in order to characterize neurosecretory cells growing in vitro. Individual pars intercerebralis could be removed free of surrounding tissue and dissociated by mechanical treatment. Mature neurosecretory neurons of different sizes regenerate new neurites during the initial three days in vitro in serum-free medium. They show a tendency to sprout one primary neurite from which fine processes develop. By means of electron microscopy, we observed the integrity of the cellular organelles, indicating that cultured neurons are healthy, and we were able to distinguish three types of neurosecretory neurons on the basis of the ultrastructural aspects of the neurosecretory material. These three types have the same ultrastructural characteristics as in situ neuroparsin, ovary maturing parsin and locust insulin related peptide neurons. Immunogold labelling at the electron microscopic level, using the two available specific antibodies, anti-neuroparsin and anti-ovary maturing parsin, confirms the morphological characterization of neuroparsin and ovary maturing parsin cells. These results show for the first time that cultured locust neurosecretory neurons behave like those in vivo, in terms of their ultrastructure and immunocytochemistry. Moreover, the presence of recently-formed neurosecretory material both in the Golgi zone of the perikaryon and in the neuronal processes indicates that cultured neurons have functional capacity since they are able to synthesize de novo and to transport the neurosecretory material along the neurite. Thus our well-characterized culture system provides a suitable in vitro model to investigate the secretory mechanism of locust neurosecretory neurons.

[Immunological similarities between two cerebral substances from Schistocerca gregaria and two neurohormones from Locusta migratoria]. / Similitudes immunologiques entre les substances cérébrales de Schistocerca gregaria et deux neurohormones de Locusta migratoria.

Two neurohormones produced by two distinctive neurosecretory median cells (A1, B) of the protocerebum have been characterized in Locusta migratoria; on with a multiple functions one called neuroparsin and one stimulating the ovarian maturation called lom OMP. Using the specific immunoserum of these two neurohormones in Schistocerca gregaria, we could demonstrate the occurring of molecules related to the neuroparsin and lom OMP of Locusta. Through histology studies of the brain and corpora cardiaca complex, the immunoserum revealed the presence of the two types of these neurosecretory cells suggesting the occurring in Locusta as well as Schistocerca of the same cells releasing molecules immunologically apparented to neuroparsin and lom OMP. The results were confirmed by electrophoretic separation of corpora cardiaca extract under unreduced conditions followed by a transfer on immobilon membrane.

Immunohistochemical investigation of locust neuroparsin-like substances in several insects, in some other invertebrates, and vertebrates.

Anti-neuroparsin serum was immunohistochemically tested on brain and/or neurohemal organ from 40 insect species belonging to 13 orders, and from 8 non-insect invertebrate and 5 vertebrate representatives using the peroxidase-antiperoxidase procedure. In insects, immunostaining of only the A1 type of the protocerebral median neurosecretory cells was revealed in all species tested of Odonata, Dictyoptera, Isoptera and Orthoptera and in 2 species from the 9 other orders out of 13 orders tested. No immunostaining was detected in vertebrate and non-insect invertebrate species except in 2 annelid species out of 4 tested. The distribution of neuroparsin-like products in Coelomata appears to be restricted mainly to 4 phylogenetically close insect orders.

Characterization of a new neurosecretory cell type containing gastrin-cholecystokinin-like peptide in the locust pars intercerebralis: ultrastructural and immunocytochemical studies, in situ and in vitro.

A new neurosecretory cell type of the locust pars intercerebralis, immunolabelled with an antiserum against a vertebrate peptide related to gastrin-cholecystokinin (CCK-8(s)), was characterized both in situ and in primary cell cultures. Semithin sections of pars intercerebralis were first immunostained in order to identify neurosecretory cells containing CCK-like material and then examined by electron microscopy. The neurosecretory cells containing CCK-like material were paraldehyde fuchsin negative and were unequivocally identified in ultrathin sections adjacent to immunostained semithin sections. They exhibited neurosecretory vesicles of variable electron density, ranging in diameter from 150 to 250 nm. Immunogold labelled ultrathin sections adjacent to unlabelled ultrathin sections allowed for the unambiguous localization of CCK-like immunoreactive material over the neurosecretory vesicles of the cells containing CCK-like material. Immunoreactivity towards CCK-8(s)-like peptide could also be detected in pars intercerebralis neurosecretory neurons grown in vitro. The CCK-like positive neurons showed a multipolar morphology with fine processes radiating from the cell body. The positive cells had the same ultrastructural characteristics as the in situ CCK-like neurons. The pattern of neurite outgrowth on reactive CCK-like neurosecretory cells in vitro and the neuroanatomical pathway of the CCK-like immunoreactive neurosecretory cells in situ could be correlated. On the basis of their number, size and localization in the locust pars intercerebralis, it is possible that the CCK-like neurosecretory cells correspond to neurosecretory cell type C, which has not, to date, been identified at the ultrastructural level.

Immunogold detection of two neurohormones: the locust ovary maturing parsin and neuroparsin.

The cellular localization of two neurohormones of the locust pars intercerebralis-corpora cardiaca system: the ovary maturing parsin and neuroparsin, was investigated using electron microscopic immunocytochemistry (post-embedding immunogold labelling). The ovary maturing parsin and neuroparsin containing cells were first identified in semithin sections treated by combined histochemical- and immunostaining. The neuroparsin cells were paraldehyde fuchsin positive (A-type cells) and the ovary maturing parsin cells were paraldehyde fuchsin negative when semithin sections were stained with paraldehyde fuchsin and immunostained with anti-ovary maturing parsin serum. The ovary maturing parsin and neuroparsin producing cells were identified on immunogold labelled ultrathin sections adjacent to double stained semithin sections. Ovary maturing parsin cells have larger more numerous vesicles of greater electron density than neuroparsin cells. The neuroparsin cells contained more lysosomal structures than the ovary maturing parsin cells suggesting different neurosecretory dynamics. Thus, immunogold labelling with antisera specific for each neurohormone demonstrates the individual nature of these two neurosecretory cells in the pars intercerebralis of the Locust.

Production and characterization of antibodies to neuroparsins A and B isolated from the corpora cardiaca of the locust.

Antisera were raised in rabbits against neuroparsins A and B which were purified to near homogeneity using electroelution from 7.5% polyacrylamide electrophoresis gels. They were characterized using immunohistochemical, enzyme-linked immunosorbent assay (ELISA), and protein-blotting methods. The antineuroparsin A and the antineuroparsin B sera have different titers and sensitivities (higher titer for antineuroparsin A, higher sensitivity for antineuroparsin B). They exhibit very good specificity. The immunohistochemical study of the entire central nervous system using either antineuroparsin A or antineuroparsin B sera shows that only the A1 type of the protocerebral median neurosecretory cells are immunostained. Moreover, among the numerous proteins of the median region of the brain and of the corpora cardiaca, each immune serum recognized only neuroparsin A or neuroparsin B. Displacement curves obtained for each immune serum by competition between either neuroparsin A or neuroparsin B demonstrate that the neuroparsin A is recognized as well as neuroparsin B, with both antisera supporting the concept that these two proteins are chemically related. The nonspecific binding of neuroparsins to an antisomatostatin immune serum used at 1/100 dilution indicates that any cross-reactivities of invertebrate molecules obtained with very low dilutions of antisera to vertebrate molecules must be considered carefully before concluding any immunological relation between invertebrate and vertebrate products.

Immunohistochemical localization of gastrin-cholecystokinin-like material in the central nervous system of the migratory locust.

Brain, corpora cardiaca (CC)-corpora allata (CA) complex, suboesophageal ganglion, thoracic and abdominal ganglia of adults, larvae and embryos of Locusta migratoria have been immunohistochemically screened for gastrin cholecystokinin (CCK-8(s]-like material. In adult, numerous immunoreactive neurons and nerve fibres are located, with a marked symmetry, in various parts of the brain and throughout the ventral nerve cord. In the median part of the brain, cell bodies belonging neither to cellular type A1 nor A2 (following Victoria blue-paraldehyde fuchsin staining) are immunopositive; their processes terminate in the upper protocerebral neuropile. In lateral parts of the brain, external cell bodies send axons into CC and some up to CA, other internal have processes which terminate in the neuropile of the brain. Two of these latter cells react also with methionine-enkephalin antiserum. In the ventral nerve cord, in addition to numerous perikarya, immunoreactive arborizations terminate in the neuropile or in close association with the sheath, at the dorsal part of all ganglia. This CCK-8(s) distribution pattern is observed only at the two last larval instars, but is precociously detected in the abdominal nerve cord of embryos, one day before hatching.

Gastrin-cholecystokinin-like and neuroparsin-like immunoreactivities in the brain and retrocerebral neuroendocrine complex of the cockroach Blattella germanica.

Using single and double labelling techniques respectively, brain-corpora cardiaca-corpora allata complexes of the cockroach Blattella germanica have been immunohistochemically investigated with antisera raised against either the vertebrate peptide gastrin-cholecystokinin (CCK-8(s] and/or the locust neurohormone neuroparsin (NPA). Single immunolabelling with anti-CCK-8(s) reveals immunoreactive perikarya and processes in median and lateral parts of protocerebrum, optic lobes, deutocerebrum and tritocerebrum. Some fibres originating in median and lateral protocerebrum are intrinsic to the brain, whereas others terminate in the nervous areas of the corpora cardiaca. Single immunolabelling with anti-NPA reveals immunoreactive cell bodies in the median part of the protocerebrum and their processes terminate both in the nervous area of the corpora cardiaca and between the intrinsic secretory cells of this neurohaemal organ. Double immunolabelling with anti-CCK-8(s) and anti-NPA enables a description of the anatomical relations between the processes and the endings of these two neurosecretory systems.

Restricted occurrence of Locusta migratoria ovary maturing parsin in the brain-corpora cardiaca complex of various insect species.

Ovary maturing parsin (OMP) is a gonadotrophic molecule previously isolated from the neurosecretory lobes of the corpora cardiaca of Locusta migratoria (acridian Orthoptera). A polyclonal antiserum directed against the two biologically active domains of the L. migratoria (Lom) OMP was used to investigate the occurrence of Lom OMP-like substances in brain-corpora cardiaca complexes of other insect species. Using immunohistochemistry, specimens of 40 different insect species belonging to 13 insect orders were tested. The Lom OMP-like substance was strictly limited to specimens of insect species belonging to the Acridae. It occurred in non-basophilic cells of the pars intercerebralis that project to the corpora cardiaca, as in Locusta. Although the antiserum only detected Lom OMP-like material in the Acridae, it is possible that related molecules exist in other insects. The antiserum may be very specific for domains of the Lom OMP molecule that have not been highly conserved during evolution or possibly these domains are not accessible to the antiserum in other insects.

Tryptophanyl-tRNA synthetase-like immunoreactivity in the central nervous system and midgut of the migratory locust. Comparisons with gastrin-cholecystokinin-like and octopamine-like immunoreactivity.

A tryptophanyl-tRNA synthetase (TrpRS)-immunoreactivity is localized in various neurosecretory cells of all ganglia of the central nervous system of the Orthoptera Locusta migratoria, except in deutocerebrum, and in endocrine cells of the midgut. It has been observed that TrpRS-like material never co-localizes either with CCK-like or octopamine-like material. TrpRS immunoreactive perikarya and processes that ramify extensively throughout the neuropiles have been detected in the protocerebrum, optic lobes, tritocerebrum, suboesophageal, thoracic and abdominal ganglia. In the lateral protocerebrum, a particular TrpRS pathway different from the lateral gastrin cholecystokinin (CCK-8(s] pathway is revealed, certain of these processes terminating in the glandular part of the corpora cardiaca. In the metathoracic ganglion, have been observed numerous immunoreactive cell bodies and processes in the neuropiles. Some of them constitute a major pathway and which are distinct from octopamine (OA) cells but in close vicinity with the latter. In the midgut immunopositive TrpRS-like cells are dispersed among the regenerative and digestive cells of the epithelium; they are different from gastrin-cholecystokinin positive cells. The various TrpRS-like immunoreactivities identified in Locusta indicate that TrpRS-like material may occur in different tissues of organisms other than Vertebrates. These results suggest also that TrpRS-like enzyme could be involved in functions other than aminoacylation, as in Vertebrates.