The dark-colour-inducing neurohormone of locusts in relation to an albino mutant of Schistocerca gregaria

In the albino mutant of an Okinawa strain of Locusta migratoria (L.) (Orthoptera: Acrididae), albinism is caused by the absence of the dark‐colour‐inducing neurohormone (DCIN), which is present in the corpora cardiaca (CC) of normally coloured phenotypes. This study tests whether the absence of DCIN is responsible for albinism in an albino mutant of another locust, Schistocerca gregaria (Forsk.) (Orthoptera: Acrididae). This seemed feasible because a single Mendelian unit controls albinism in both species. However, implantation of CC, or injection of an extract of CC, from albino donors of S. gregaria, induce dark coloration in crowded nymph recipients of the Okinawa albino mutant of L. migratoria, as effectively as do implanted CC, or injections of extract of CC, from normal phenotype donors of S. gregaria. Therefore, DCIN is present in the albino mutant of S. gregaria, and consequently, the albinism in this mutant is not caused by its absence. Implantation of CC, or injection of extracts of CC, from albino donors of S. gregaria to conspecific albino nymphs does not induce darkening. Only extremely high doses of synthetic DCIN injected into albino nymphs of S. gregaria are effective, inducing some darkening. The dose to induce such darkening in albino nymphs of S. gregaria is 50 nmol, ≈ 5 × 10⁶ times higher than that (10 femtomol) needed to induce equivalent darkening in nymphs of the Okinawa albinos of L. migratoria. The results are discussed and some possible explanations of the observed effects outlined.     

Immunocytochemical and ultrastructural study of the frog (Rana pipiens) pars distalis with special reference to folliculo-stellate cell function during in vitro superfusion

Summary The colloidal gold immunocytochemical technique was used to determine the ultrastructural features of the glandular cells in the pituitaries of male frogs, Rana pipiens, both in vivo and after superfusion in vitro. Specific reactions to antisera against bullfrog gonadotropins, human prolactin, and synthetic 1–39 corticotropin allowed identification of the 3 corresponding types of glandular cells. No immunoreaction was obtained with antisera against human or ovine-growth hormone, human -thyrotropin hormone, and bovine S-100 protein. General morphological features of these immunocytochemically identified glandular cells were similar to those of equivalent cells previously described in other amphibian species. Non-glandular folliculo-stellate cells were distinctive. In freshly removed pituitaries, these folliculo-stellate cells contained lysosome-like structures, but did not show phagocytic vacuoles in the cytoplasm; they contained many mitochondria, and the Golgi complex and endoplasmic reticulum were relatively undeveloped. After 4 or 18 h of superfusion, some immunoreactive gonadotropic, prolactin, and corticotropic cells showed degeneration and destruction. In the same gland, folliculo-stellate cells retained a viable appearance, but showed phagocytic vacuoles containing secretory granule-like structures which were immunoreactive to gonadotropic, prolactin, and corticotropic antibodies. Some folliculo-stellate cells showed phagocytic vacuoles containing complete glandular cells. These results suggest that superfusion causes a destruction of some of the glandular cells, and that folliculo-stellate cells act as phagocytes when cellular debris or moribund cells are present in the intercellular space in the pituitary parenchyma.Supported by grant DCB 8710462 from the National Science Foundation, grant 2148-83 from the CAICYT (Spain) and the Junta de Andalucia (Spain)     

The levels of ecdysteroids in uninjured and leg-autotomized nymphs of Blattella germanica (L.)

The levels of ecdysteroids in control and leg-autotomized first-instar nymphs of Blattella germanica were determined by radioimmunoassay from hatching to the time of the first ecdysis. Uninjured nymphs showed a distinct release of ecdysteroids half-way through the stadium, and this resulted in the commencement of the moult cycle which formed the cuticle of the second instar. Cockroaches which had legs autotomized at 48 h after hatching (i.e. before the control ecydsteroid release) had their instar duration increased by that time period. Releases of ecdysteroids and events of the moulting cycle were also postponed by the 48 h period. The titre of ecdysteroids in injured animals was double that of controls. Nymphs were also autotomized at 96 h (i.e. after the normal release of ecdysteroids) but no changes in instar duration, ecdysteroid releases, or events of the moult cycle were recorded. The effects of injury, prothoracicotropic hormone activity and ecdysteroid release are discussed.     

Spatial and temporal distribution of pheromone biosynthesis-activating neuropeptide in Helicoverpa (Heliothis) armigera using RIA and in vitro bioassay.

A [3H]-PBAN (pheromone biosynthesis-activating neuropeptide) analog was synthesized, and binding of the radioligand to a specific PBAN-antiserum was achieved. The inhibition of binding of the radioligand by unlabeled PBAN, several PBAN analogs, and other competitors was studied and a specific radio-immunoassay was developed. Using this radioimmunoassay we found PBAN-like immunoreactivity in methanol extracts of hemolymph and neural tissues from females. Higher levels of PBAN-like immunoreactivity in extracts of brain-suboesophageal ganglion complexes, corpora cardiaca, thoracic ganglia, and abdominal ganglia were observed during the 4-5th h scotophase when compared to the PBAN-like immunoactivity levels during the 6-11th h photophase. On the other hand, the concentrations of PBAN-like immunoreactivity, in the terminal abdominal ganglion were higher during the photophase relative to minimal levels observed during the scotophase, indicating an accumulation before the onset of pheromone production. These differences in concentrations of PBAN were also reflected in the stimulation of in vitro pheromone glands, whereby significant stimulations were obtained by scotophase and photophase brain extracts, scotophase thoracic ganglia extracts, and photophase terminal abdominal ganglia extracts. No detectable levels of PBAN were found in hemolymph extracts during the sampling periods.     

Evidence for both humoral and neural regulation of sex pheromone biosynthesis in Spodoptera littoralis

Selected tissues presumably involved in the control of sex pheromone production were analyzed by ELISA for the presence of PBAN-like immunoreactivity (PBAN-IR) in Spodoptera littoralis. The temporal distribution pattern of PBAN-IR in the hemolymph is similar to that of pheromone production in the gland. On the other hand, analysis of the retrocerebral complex, brain-subesophageal ganglion complex, and terminal abdominal ganglion (TAG) revealed similar PBAN-IR levels in both photophase and scotophase periods. Pheromonotropic activity exhibited by both hemolymph and TAG, as determined by a modified in vitro bioassay, agrees with the results of the immunochemical analyses. Severing the ventral nerve cord anterior to the TAG impaired normal sex pheromone production by second-scotophase females. These results are discussed in the context of how sex pheromone biosynthesis is regulated by PBAN in S. littoralis. © 1996 Wiley-Liss, Inc.     

Comparison of the Protection against Neuronal Injury by Hypothalamic Peptides and by Dexamethasone

The comparative study has been carried out on hypothalamic neurohormone (proline-rich polypeptides-PRP) and synthetic glucocorticoid dexamethasone (DEX) protective properties at the systemic (i/m) administration. Both background and evoked electrical activity (on n.ischiadicus stimulation) of single neurons in the lumbo-sacral part (laminae II–VI and VII–VIII by Rexed) and field potentials (FP) of spinal cord were recorded during acute experiments on intact spinal rats, subjected to Vipera Raddei (VR) venom intoxication, and chronic spinal cord trauma (hemisection). The action of PRP was characterized by the pronounced activation of the background activity (BA) with adaptive effect, depending on dose and initial level of BA, by results of the statistical analysis. A high effect is received from comparatively small doses. For comparison it was used strong glucocorticoid DEX, possessing single-directed but less expressed excitative action on investigated spinal cord neurons. The initial increase of BA frequency with subsequent depression was the typical symptom of venom influence. A protective effect of preliminary PRP injection is revealed on the succeeding VR venom influence. Use of PRP and DEX causes the increase of reduced activity of neurons on the injury side of animals with spinal cord hemisection. It provides the possibility of the therapeutic utilization. It was revealed considerably more expressed PRP action on neurodegenerative process connected to spinal cord injury (in comparison with DEX). The influence of hormones was compared in identical conditions of experiments on non-injured (control) and injured sides. Taking into consideration revealed protection characteristic of PRP and also the ability of snake venom to stabilize and to prolong its action combined with these preparations, the assumption is made on prospective use of the specified combination in clinical practice.     

Protection Against Snake Venom-Induced Neuronal Injury by the New Hypothalamic Neurohormone

The action of PRP is characterized by the pronounced activation of the background activity (BA) of the brain spinal cord, and the degree of the activity depends on BA initial level. The typical peculiarity of Vipera raddei venom influence is the initial increase in frequency of BA with subsequent depression. A preliminary injection of PRP has a protective effect at subsequent influence of venom. In animals with hemisection the PRP increases the decreased activity of neurons on injury side. Taking into consideration the protective peculiarities of PRP in the relationship to snake venom and the possibility of the latter to stabilize and prolong the action of drugs (in the case of PRP) combined with them, it is supposed that the mentioned use of the combination in clinical practice will be perspective. The data obtained testify the PRP to be a neuroprotector against many toxic compounds formed in organism (glutamate, ceramid, beta-amyloid neurotoxisity, etc.). Investigations in this aspect are still in the process.     

Somatostatin-14 influences pituitary–ovarian axis in peripubertal rats

The effects of multiple somatostatin (SRIH-14) administration on the pituitary-ovarian axis were examined in peripubertal rats. Female Wistar rats received subcutaneously, two daily doses of 20 mug SRIH-14 per 100 g body weight (b.w.) for five consecutive days (from the 33rd to the 37th day of life). Follicle-stimulating (FSH), luteinizing (LH) and somatotropic (GH) cells were examined by the peroxidase-anti-peroxidase immunocytochemical method. Changes in cell volumes, volume densities and number per unit area (mm(2)) of FSH-, LH- and GH-immunoreactive cells were evaluated by stereology and morphometry. Serum FSH and LH levels were determined by RIA. Ovaries were analyzed by simple point counting of follicles. The volumes and volume densities of FSH-, LH- and GH-immunoreactive cells were significantly decreased while their numbers per mm(2) remained unchanged. SRIH-14 induced a significant decrease in serum FSH and LH levels. In the ovary, SRIH-14 induced an increase in the number of primordial follicles, followed by a reduction in the number of small healthy growing follicles and absence of preovulatory follicles. The number of atretic follicles was unchanged. We concluded that treatment with SRIH-14 during the peripubertal period markedly inhibited pituitary FSH, LH and GH cells. In the ovary, SRIH-14 acted by inhibiting folliculogenesis without affecting atretic processes.     

Effect of salmon gonadotropic hormone on sex steroids in male rainbow trout: plasma levels and testicular secretion in vitro

Summary Male rainbow trout were treated with salmon gonadotropic hormone (GTH) at different stages of the circannual reproductive cycle; spawning fish were also treated with an antiserum against salmon GTH. Injection of GTH led to a several-fold increase of plasma sex steroid levels during spermatogenesis and in the spawning season but was without effect at early stages of testicular development. GTH neutralization during the spawning season was followed by a several-fold decrease of plasma sex steroid levels. During spermatogenesis and in the spawning season, both treatment regimes resulted in an increased sensitivity of testicular explants in response to a subsequent stimulation of steroid secretion in vitro. This up-regulatory response may facilitate and maintain the high sex steroid plasma levels observed during the spawning season. It may also be necessary to allow for concomitant peak values of plasma GTH and sex steroids in the spawning season, a situation difficult to understand within the negative feedback concept. The adaptive capacities of the testicular steroidogenic system indicate that it is not only an effector site for GTH but also an active part of the endocrine system controling reproduction.Abbreviations BSA bovine serum albumin - bw body weight - E₂ 17-estradiol - GnRH gonadotropin releasing-hormone - GTH gonadotropic hormone - LH luteinizing hormone - OHT 11-hydroxytestosterone - OT 11-ketotestosterone - 17-20P 17-hydroxy, 20-dihydroprogesterone - PE pituitary extract - raGTH rabbit anti-GTH antiserum - rPS rabbit preimmune serum - T testosterone     

Production of antibodies to Manduca sexta cerebral neurosecretory cells

Understanding the neuroendocrine regulation of insect development depends upon having antibody probes to the neurohormones involved, which are usually present at trace levels, making antibody generation difficult. This report describes a simple method for producing antibodies specific to cerebral neurosecretory cells (NSC) of the tobacco hornworm, Manduca sexta, and to the neurohormone(s) they produce. The method involves the isolation of only a few hundred NSC somata (～ 0.3 μg of protein) that serve as the immunogen. The cerebral NSC used were the L-NSC III, the prothoracicotropes, that produce the prothoracicotropic hormone (PTTH), the principal neuroendocrine effector of insect molting and metamorphosis. A PTTH-containing extract of microsurgically isolated somata of the L-NSC III was injected intraperitoneally into a Balb/c mouse. The antiserum produced specifically immunostained the L-NSC III in wholemounts of brains from different developmental stages. This antiserum also contained antibodies directed against PTTH, as shown by its ability to inhibit the neurohormone's biological activity in an in vitro prothoracic gland bioassay. Such antiserum can be used to investigate the ontogeny and phylogeny of NSCs. With the hybridoma technique, monoclonal antibodies to individual NSC proteins (PTTH) could be obtained, circumventing the need to purify them for antibody production. This method should be applicable to comparable neurosecretory cell systems in other insect species.