Control of ovarian steroidogenesis in insects: a locust neurohormone is active in vitro on blowfly ovaries.

Ovarian steroidogenesis controlling insect reproduction is mainly regulated by brain gonadotropins liberated from corpora cardiaca (CC). Till now, different neurohormones have been identified in two insect groups only, locusts and mosquitoes, and it is unknown whether they could be active in other insects. In order to complete previous observations on the control of ovarian steroidogenesis in the blowfly, Phormia regina, we examined whether neuropeptides isolated from locust CC have an effect in vitro on ovarian steroidogenesis in our dipteran model. Our experiments showed that crude extracts from locust CC efficiently stimulated steroidogenesis in blowfly isolated previtellogenic ovaries. However, such an activity was observed neither with authenticated neuroparsins (NPs), the putative homologs of the ovarian ecdysteroidogenic hormone of mosquitoes, nor with ovarian maturing peptide (OMP), the putative locust steroidogenic neurohormone. Partial purifications of CC extracts were then performed using methanol and/or acidic ethanol extractions followed by reverse phase HPLC and collected fractions were assayed in vitro. A significant steroidogenic activity was found in a single group of acidic fractions, well separated from OMP and NPs, which was associated to slight but significant anti-insulin immunoreactivity. In conclusion, a locust CC neurohormone, different from NPs and OMP, is able to stimulate ecdysteroidogenesis in blowfly ovaries. Though this active factor has not been fully characterized, its behavior during extraction or HPLC and its immunoreactivity strongly suggest it could be an insulin-like peptide. This is in agreement with previous studies demonstrating the role of such peptides as steroidogenic gonadotropins in blowflies and several other insects.

Control of ovarian steroidogenesis by insulin-like peptides in the blowfly (Phormia regina).

This study investigated the ability of insulin and of insect insulin-like peptides (ILPs) to stimulate ovarian steroidogenesis in the blowfly Phormia regina. Bovine insulin was active on ovaries isolated in vitro, which showed an age-dependent sensitivity; this peptide progressively stimulated steroidogenesis in ovaries isolated from the third day after adult molt, but not in younger ones, and had maximal activity after the fifth day. This stimulatory effect was observed equally from females reared in the presence or in the absence of males, excluding a regulatory effect of mating. The mode of action of insulin in blowflies did not involve cAMP, but triggered a specific and well-conserved transduction cascade. In particular, a peroxovanadium compound, known to activate specifically the insulin receptor in mammals, also stimulated blowfly ovarian steroidogenesis in vitro. Conversely, chemicals known to inhibit the mammalian insulin receptor or downstream elements of its signaling pathway, such as LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), were able to prevent the steroidogenic action of bovine insulin on fly ovaries. Extracts from the median neurosecretory cells (MNCs) of blowfly brains, which are known to contain endogenous ILPs, stimulated ovarian steroidogenesis very efficiently and were also sensitive to inhibition by LY294002. These experiments indicated the involvement of PI3K in the mode of action of MNC extracts and substantiated that their endogenous ILPs are involved in the regulation of ovarian steroidogenesis. This conclusion was corroborated by the effects of synthetic bombyxin II, an ILP originating from silkworm MNCs, which also stimulated steroidogenesis in isolated blowfly ovaries. Altogether, these data suggest that insulinlike neurohormones from MNCs play a crucial role as steroidogenic gonadotropins in female flies.

Inhibition of ovarian steroidogenesis by cyclic GMP in a fly.

Previous investigations in the female blowfly Phormia regina have shown that 3-isobutyl-1-methylxanthine (IBMX), a broad spectrum inhibitor of phosphodiesterases (PDEs), fails to mimic the steroidogenic effects of cAMP on ovaries, although it efficiently increases the concentrations of this second messenger. In this study, experiments carried out to clear up this contradiction demonstrated that IBMX, besides its effect on cAMP, also increased cGMP concentrations in blowfly ovary and that these two cyclic nucleotides controlled ovarian steroidogenesis antagonistically. In particular, a selective inhibitor of cGMP-specific PDEs, unlike IBMX, had a very strong negative effect on ovarian steroidogenesis. Moreover, a cGMP analog was able to inhibit steroid biosynthesis in previtellogenic and vitellogenic ovaries, thus affecting basal and acute steroidogenesis respectively. Our observations also demonstrated that cGMP was always present in blowfly ovary, reaching its maximal levels at the end of vitellogenesis, in close correlation with the physiological decrease in ovarian steroidogenesis. Experiments using an inhibitor of protein kinase G clearly indicated that the effects of cGMP were mediated by this enzyme. On the contrary, these effects did not seem to involve cGMP-regulated PDEs or ion channels. Our results also indicated that ovarian cGMP concentrations were not controlled by brain factors, suggesting a probable involvement of paracrine/autocrine factors. Nitric oxide (NO) appeared to be a good candidate for such a control, because an NO donor was able to stimulate ovarian cGMP concentrations and to drastically decrease ovarian ecdysteroid biosynthesis in blowflies. These data thus demonstrate, for the first time in invertebrates, a potent role of cGMP in the negative control of ovarian steroidogenesis and suggest a possible co-regulation with NO.

Calcium inhibits ovarian steroidogenesis in the blowfly Phormia regina.

Calcium is frequently involved in the stimulation of steroidogenesis in gonads and endocrine glands, generally in association with cAMP. However, our present observations show that it has the opposite effect in the ovary of the blowfly Phormia regina. Our in vitro experiments first showed that extracellular calcium does not play a role during the stimulation of steroidogenesis in fly ovaries; indeed steroidogenesis was activated in vitro as efficiently in a medium with or without calcium, either by pharmacological compounds mimicking cAMP signaling or by active brain extracts. When calcium was experimentally introduced into biosynthetic cells by ionophores or liberated from internal stores by thapsigargin, it did not activate, but clearly inhibited both basal and acute steroidogenesis respectively in previtellogenic and in vitellogenic ovaries. Our experiments also demonstrated that calcium decreases cAMP concentrations in the ovaries of Phormia, by stimulating its degradation, without modifying its biosynthesis. Moreover, inhibitors of calcium-calmodulin phosphodiesterases (PDEs) increased steroid biosynthesis in vitro, whereas inhibitors of calcium-insensitive PDEs did not. These data thus demonstrate that, in blowfly ovaries, calcium ions inhibit cAMP-stimulated steroidogenesis by activating a calmodulin-sensitive (type I) PDE.

Cyclic AMP-dependent and independent stimulations of ovarian steroidogenesis by brain factors in the blowfly, Phormia regina.

The involvement of cyclic-AMP (cAMP) as a potential second messenger in the neurohormonal control of ovarian steroidogenesis was investigated in the adult female blowfly Phormia regina. Individual measurements of ovarian cAMP concentrations and of ovarian biosynthesis of ecdysteroids, stimulated after a protein meal, demonstrated that steroidogenesis is preceded by a peak of cAMP in the ovaries. In vitro, ovarian steroidogenesis was stimulated by cell-permeable analogues of cAMP and by forskolin. Crude brain extracts were also able to elicit a rise of cAMP in the ovaries in vitro and the secretion of ecdysteroids into the medium: such extracts were more active before than after the protein meal, suggesting a rapid release of neuroendocrine material after feeding. Extracts were then prepared from the dorso-medial part of the brain, containing the neurosecretory cells of the pars intercerebralis (PI): these extracts were again found to stimulate the ovarian ecdysteroid secretion, but surprisingly, they failed to trigger a rise of cAMP in the ovaries in vitro. However, extracts from the rest of the cephalic nervous mass, deprived of PI, were also steroidogenic and they increased ovarian cAMP. Experiments with Rp-cAMPS, a cAMP antagonist, were not able to prevent the ecdysteroid stimulation by PI extracts, but did so partly for the extracts deprived of PI. This study thus indicates that at least two different cephalic factors are able to stimulate ovarian steroidogenesis in the blowfly, one elaborated by PI and acting via a cAMP-independent mechanism, and the other elaborated outside PI and using cAMP as a second messenger.

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.

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.

Detection of putative dopamine receptors in neurites outgrowing from locust central nervous system explants using anti-idiotypic dopamine antibodies.

Organotypic cultures established from the third thoracic ganglion of locust embryo have been used to investigate dopamine receptors. In this in vitro system, neurites emerge directly from the explants and form a dense network around the explants, presenting cell surface freely exposed for experimental labelling. Polyclonal anti-idiotypic antibodies raised in rabbits to antibodies against dopamine conjugate, and previously found to bind to dopamine receptors, have been used to investigate putative dopamine receptors in these neurites. Immunocytochemical detection by light microscopy employing immunofluorescence labelling, was correlated with electron microscopy, using peroxidase staining. In addition to a location for dopamine receptors on the neurite surface, intracellular binding sites were also found in neurites. This internal labelling might represent an intracellular pool of dopamine receptor precursors. The labelling was specific in that it was not present when the anti-idiotypic dopamine antibodies were replaced with non-immune serum or when preincubation with conjugated dopamine preceded incubation with anti-idiotypic dopamine antibodies.

In vitro growth of locust embryonic pars intercerebralis neurosecretory cells.

Neurosecretory brain cells from embryonic locusts cultured in serum-free medium failed to show any visible signs of growth. In contrast, the same neurons co-cultured with CNS explants (brain-retrocerebral complexes and thoracic ganglia) show excellent axonal growth: sprouting occurs after one day of co-culture and increases within the first week. These results indicate the production of an active neurite outgrowth stimulating factor by co-cultured CNS explants. The similarity of the stimulating effects by the two explants on neurite outgrowth rule out brain neurohormones as probable candidates for the stimulating factor. In addition, neither insulin nor neuroparsin added to the culture medium to test their trophic effect improves the growth of the cells. Conditioned medium derived from cultures of brain-retrocerebral complexes produced no neurite outgrowth, suggesting that the active factor released in the medium by brain explants does not remain free in solution but binds to the substratum. Finally, neurons co-cultured with CNS explants attached to the bottom of the culture dish develop neurites only when in close proximity to the explants. This observation strongly suggests the binding of an active neurite outgrowth stimulating factor to the substratum in the vicinity of the explants. As a control for CNS explants, the action of non-nervous tissue was tested: a similar, but less extensive neurotrophic effect, was observed with esophagus segments co-cultured with neurosecretory brain cells. These results demonstrate that locust neurosecretory neurons isolated in cell culture require combined explants for elaborating processes and suggest that the neurite promoting effect is mediated by a substrate-associated molecule(s).(ABSTRACT TRUNCATED AT 250 WORDS)

Differentiation of glial cells and neurite outgrowth obtained from embryonic locust central nervous system explants.

Up to the present it has not been possible to obtain viable glial cells from dissociated insect nervous system cultures. We report here that the use of explant culture of locust embryo central nervous system (CNS) has been successful in allowing the proliferation of glial cells derived from glial precursors located at the periphery of the embryonic CNS. In such cultures, maintained for 3 months under specific conditions, 4 cell types at intermediate stages of differentiation can be distinguished around the explants after 2 weeks in vitro. They have been identified by scanning and transmission electron microscopy. The first type (stage 1) consists of flat epithelioid cells with an abundant and lucent cytoplasm containing little granular endoplasmic reticulum. These earliest cells subsequently develop flat ameboid prolongations forming an intermediate cell type (stage 2) which then differentiates into protuberant bipolar cells (stage 3) in which appear well-organized cisterns of granular endoplasmic reticulum. The last stage of differentiation (stage 4) is composed of multipolar cells with an electron-dense cytoplasm and well-defined processes characterized by the presence of ribosomes and granular endoplasmic reticulum. These (stage 4) differentiated cells resemble mature glial cells. In the same in vitro system, neurites, growing from neurons originating in the explants, form a network lying upon the glial cells and in close relationship with them. Neurites present growth cones and lack ribosomes and granular endoplasmic reticulum. We conclude that this model is a potentially useful system for use in in vitro studies of insect glia and neurite-glia interactions.

An in vitro autoradiographic study of gliogenesis in the embryonic locust brain.

The origin of glial cells in the embryonic locust brain was studied by in vitro labeling with tritiated thymidine during the second half of embryonic life from the 6th day (E6) to hatching (E12). Autoradiographic analysis revealed a germinal zone surrounding the brain. Until the 9th day of embryonic age (E9) this zone consisted of 26 clusters of mitotically active undifferentiated cells. From E10 to E12 only 14 undifferentiated cell clusters were found and, moreover, the labeling declined considerably in the remaining clusters. Clusters were always located at the level of bundles of fibers and labeled cells were present along these fibrous tracts. As early as E6 glial cells were labeled including the glial cells located in the rind between the neurons and the glial cells surrounding the neuropil. The pattern of glial cell labeling was similar to that of the undifferentiated cell clusters, reaching a peak between E6 and E9 then declining. On the basis of these findings it is concluded that glial cells of the developing brain of Locusta migratoria derive from precursor cells located in a peripheral germinal zone and it is suggested that the cells formed utilize the bundles of fibers as a migratory pathway to reach their destination in the brain. Furthermore, this study indicates that the time of appearance of glial cells coincides with that of neuronal maturation.

Undifferentiated cells present in the pars intercerebralis of larval and adult locusts are glial precursors. Autoradiographic and ultrastructural study in vivo and in vitro.

The intercerebral part of the protocerebrum from embryos, larvae and imagos of Locusta migratoria was investigated in vivo and after culture of the brain in vitro using light and electron microscopy. The results showed the presence in embryo and persistence in larva and adult of 2 clusters of mitotically active embryonic cells in the inner part of each half of the pars intercerebralis. The fate of these undifferentiated cells was investigated during postembryonic life by in vitro and in vivo labeling with tritiated thymidine combined with counts of nervous cells of the pars intercerebralis. Autoradiographic results confirmed the mitotic activity of the undifferentiated cells and established the pattern of this activity which declines from the third larval instar to adult stage. Mitoses were never seen in neurons and glial cells. Neurons were unlabeled and their number was constant. Glial cells were labeled and their number increases throughout postembryonic life with a pattern of proliferation similar to the pattern of mitotic activity of the undifferentiated cells. These observations indicate that the undifferentiated cells of the pars intercerebralis of the locust represent a source of glial cells and could be called glioblasts.

[Morphological and biochemical events in the periovulatory period in ovarian follicles of rats. In vitro studies]. / Evénements morphologiques et biochimiques liés à la période périovulatoire de folicules ovariens de rattes. Etude in vitro.

Cultures of rat ovarian follicles obtained at the proestrus were investigated during 24 hours following a supply of gonadotropins (FSH, LH, Prolactin) used in order to stimulate their physiological rise at the proestrus. As showed by the cytological study, the initiating luteinization appeared in follicles after 24 hours. Progesterone, testosterone and oestradiol-17 beta were assayed in culture media. The three gonadotropins induced a significant fall of the oestradiol-progesterone ratio whereas testosterone levels were unmodified; at first they increased and then inhibited the oestradiol-17 beta synthesis. On the other hand, progesterone rose one hour after oestradiol-17 beta but its secretion persisted. Thus, the inhibition of the enzymatic aromatization of steroids on one hand, the evidence of a new metabolic way leading to the increasing synthesis of progesterone on the other hand would be due to the preovulatory rise of gonadotropins. After exposition for 4 hours to FSH, LH and Prolactin, a new supply of LH did not modify testosterone and oestradiol-17 beta levels but led to a significant increase in the secretion of progesterone. so mechanisms induced by the proestrus preovulatory peak would seem irreversible.

[Androgen activity of dysgenesic rats' testis. Histochemical, ultrastructural studies and radioimmunoassay of testosterone]. / Activité androgène de testicules dysgénésiques de rats.

Androgen activity of rat testis submitted to a radiomimetic, Misulban, during prenatal development has been explored with different technics. Histochemical method shows the presence, in the interstitial cells, of delta5-3beta-hydroxysteroid dehydrogenase (delta5-3beta-HSD), and others enzymes of cellular metabolism, particularly the glucose-6-phosphate dehydrogenase (G6PDH) which is a glucidic enzyme implicated in the specific metabolism of steroidogenesis. From ultrastructural point of view interstitial cells contain the organels proper to steroidogenetic cells (important smooth endoplasmic reticulum, many voluminous mitochondria with tubular cristae). The plasma testosterone concentration measured by radioimmunoassay, confirms the androgenic function of interstitial tissue in dysgenesic testicles.

[Ultrastructure of testicular Sertoli cells in normal and dysgenesic rats, during the fetal and postnatal periods]. / Ultrastructure de la cellule de Sertoli de testicules de rats normaux et dysgénésiques, en période foetale et postnatale.

Electron microscopic study of Sertoli cells from normal and Misulban-treated rats has been realized during foetal and postnatal life. From 15th day of foetal life, morphological aspects of Sertoli cells plead for a steroidogen activity and protein synthesis. Ultrastructural organels of Sertoli cells are the same in sterile and normal seminiferous tubule, except the Sertoli junctions which are less numerous in sterile tubules and appear later.

[Ultrastructure of Sertoli cells in male rats treated with "misulban" and stilbestrol]. / Ultrastructure de la cellule de Sertoli chez le rat mâle soumis á l'action du "misulban" et du stilbestrol

The seminiferous tubulars of rats testis, subject to the action of Misulban during their foetal life, contain only Sertoli cells and constitute a good model for their study. From the ultrastructural point of view, these cells present several important mitochondria of tubular crest from, an abundant smooth endoplasmic reticulum, a Golgi apparatus well developed, a cytoplasm rich in ribosomes, Lipids droplets and intercellular junctions whose number increases with age. The administration of diethylstilbestrol, which inhibits gonadotropic and androgenic secretions reduces the signs of activity in the Sertoli cells.

[Ultrastructural study of dysgeneic ovaries of rats, resulting from Misulban administration during fetal life]. / Etude ultrastructurale d'ovaires dysgénésiques de rattes, obtenus par administration de misulban pendant la vie foetale

She-rats have been treated at the 15th day of gestation with Misulban. The she-rats born from the former ones present an ovarian dysgenesis due to a precocious destruction of the germinal cells by the radiomimetic. These dysgeneses are characterized by the presence of cordal epithelial structures and of stroma. Cells whose cytoplasmic infrastructure is characteristic of a steroidogenesis have been searched on ovaries taken of between 15 days and 6 months. In the absence of follicular organization, they appear only from puberty and are localized particularly, between the epithelial structures, in the conjonctive stroma.