Ultrastructural localization of egg-laying prohormone-related peptides in the atrial gland of Aplysia californica

The atrial gland is an exocrine organ that secretes into the oviduct of Aplysia californica and expresses three homologous genes belonging to the egglaying hormone gene family. Although post-translational processing of the egg-laying hormone precursor in the neuroendocrine bag cells has been examined in detail, relatively little is known about the post-translational processing of egg-laying hormone-related gene products in the atrial gland. A combination of morphologic techniques that included light-microscopic histology and immunocytochemistry, transmission electron microscopy, and immuno-electron microscopy were used to localize egg-laying hormone-related peptides in the atrial gland and to evaluate the characteristic morphology of their secretory cells. Results of these studies showed that there were at least three major types of secretory cells in the atrial gland (types 1–3). Significantly, of these three cell types, only type 1 was immunoreactive to antisera against egg-laying hormone-related precursor peptides. The immunoreactivity studies established that all three egg-laying hormone-related precursor genes are expressed in type-1 cells and indicated that the processing of these precursors also occurs within the secretory granules of this cell type. Evidence was also obtained that proteolytic processing of the egg-laying hormone-related precursors differed significantly from that observed in the bag cells. In contrast to the bag cells, the NH₂-terminal and COOH-terminal products of the egg-laying hormone-related precursors of the atrial gland were not sorted into different types of vesicles.     

Environmental temperature modulates the rate of synthesis of egg-laying hormone inAplysia

Summary Synthesis of egg-laying hormone by the neuroendocrine bag cells ofAplysia californica is approximately twofold higher in summer than in winter. To determine whether this seasonal variation in peptide synthesis is triggered by seasonal variations in ocean temperature, animals were obtained at various times of the year and held for prolonged periods at either 15 or 20°C prior to measuring hormone synthesis. Rates of synthesis in winter animals held at 20°C began to increase after two weeks, reaching a level twice that of 15°C animals by six weeks. Cooling to 15°C blocked the rise in hormone synthesis that normally occurs between May and June and reduced synthesis in summer animals to values typical of winter. It is concluded that the seasonal modulation of neurohormone synthesis is due to changes in environmental temperature.     

In vivo recordings of neuroendocrine cells (caudo-dorsal cells) in the pond snail

Summary Ovulation and egg-laying behavior in the pond snailLymnaea stagnalis are controlled by the neuroendocrine caudodorsal cells (CDCs), constituting two clusters — one in each cerebral ganglion — totaling about 100 cells. In vitro studies have shown that the CDCs release their products, including the ovulation hormone, during a burst of spiking activity lasting for about 30 min (CDC discharge). This burst can be initiated by repeated intracellular stimulation with depolarizing current pulses, in which case the firing pattern is termed afterdischarge.Using cuff electrodes we recorded extracellularly from the intercerebral commissure, (the neurohaemal area of the CDCs) to study the activity of these cells during spontaneous egg-laying of freely behaving snails.The cuff-implanted snails showed long-lasting spiking activity prior to every bout of egg-laying. These spontaneous long-lasting discharges had several features in common with the intracellularly recorded activity of the CDCs in vitro: the time courses of spike broadening and of firing rates in the cuff-implanted animals were very similar to the characteristic patterns found in the isolated brain. Firing rates were higher and durations were longer in the cuff-implanted animals, however. In vitro, the duration of the discharge could be prolonged appreciably by recording in blood instead of normal saline, indicating that the bathing fluid normally used causes shortening of the CDC discharge. The way in which CDC discharges are triggered is discussed as a possible explanation for the differences in firing rates.The pattern of locomotion during spontaneous egg-laying was largely similar in cuff-implanted and unoperated animals. The level of locomotion was lower in the experimental animals. In addition, the rate of locomotion only partially returned to pre-oviposition levels. This is ascribed to the effect of the operation.It is concluded that the afterdischarge is the natural firing pattern of the caudodorsal cells ofLymnaea, and that this firing pattern constitutes the central event in the egg-laying behavior of this animal.Abbreviations CDC caudodorsal cells - CDCH caudodorsal cell hormone - CDCA caudodorsal cell autotransmitter     

Isolated prolactin and growth hormone granules from bovine pituitary: content and stability are seasonally dependent

Secretory granules containing prolactin (PRL) and growth hormone (GH) as essentially the only proteins were isolated by centrifugation. PRL and GH varied reciprocally in the granule preparations with the seasons. During winter PRL content was lowest (20%) and GH highest (80%); during summer the converse obtained: PRL, 70% and GH,, 30%. Both hormones were in almost equal proportion during the spring. The amount of either hormone released from granules and pituitary slices was directly related to its relative content in the gland. The pattern of PRL release from secretory granules and pituitary tissue in vitro was similar to that reported for blood levels in ruminants: low during winter and high during summer. It is concluded that seasonal factors affect primarily the synthesis and/or storage of PRL and GH, and there exists a direct relationship between intracellular stores and release.     

Juvenile hormone-dependent motor activation in the adult locust <Emphasis Type="Italic">Locusta migratoria</Emphasis>

Abdominal motoneurones of the locust Locusta migratoria were investigated in immature, mature and allatectomised females to compare their response characteristics during reproductive development. These motoneurones were chosen because they control muscles which are involved in extreme lengthening during egg-laying behaviour. The study focused on changes in motoneurone firing activity and its possible regulation by juvenile hormone. In isolated nerve-muscle preparations, increased resting motor activity was found in mature (>14 days) but not in immature females (<5 days). Removing the corpora allata, the gland producing juvenile hormone in insects, prevented increased motor activity. Stimulus evoked activation of the motor system led to a characteristic burst of action potentials which lasted for a few seconds. The time-course and amount of activation changed significantly during reproductive development. Mature females displayed longer lasting and higher activity than immature or allatectomised females, but only those segments involved in egg-laying were found to express the altered firing properties. Single cell analysis of motoneurone dendritic morphology or membrane properties revealed no evidence that could be causative for the activity changes seen during reproductive development. The results suggest that altered motoneurone activity serves to adapt females to the neuromuscular requirements of egg-laying behaviour.     

Regional differences in processing of locally translated prohormone in peptidergic neurons of Aplysia californica

Earlier work showed that cell bodies and neurites of the peptidergic bag cell neurons of Aplysia californica contain mRNA for egg-laying hormone. The purpose of the present study was to determine if egg-laying hormone synthesis and prohormone processing is similar in the pleurovisceral connective nerves (containing neurites of bag cell neurons) and the bag cell neuron clusters (containing both cell bodies and neurites of bag cell neurons). Initial experiments confirmed by RT-PCR and sequencing that egg-laying hormone mRNA was present in the pleurovisceral connective nerves. To investigate possible regional differences in translation of mRNA and prohormone processing, clusters were separated from connective nerves and newly synthesized egg-laying hormone-immunoreactive proteins were analyzed. Results showed that synthesis and processing of prohormone occurred in both the clusters and isolated connective nerves; however, the relative abundance of prohormone, processing intermediates, and egg-laying hormone was different. Pulse-chase experiments showed that prohormone was processed more slowly in the connective nerves than in the clusters. These results show that mRNA in isolated neural processes of neuroendocrine cells can be translated, and that the cellular machinery for protein synthesis is present, but processing of the ELH prohormone is significantly compromised.     

GH3 cell secretion of growth hormone and prolactin increaseases spontaneously during perfifusion

Summary GH₃ cell secretory activity was studied in long-term perifusion to define previously reported spontaneous increases in growth hormone (GH) and prolactin production (PRL). Mechanically harvested cells (1×10⁷/column) were perifused at 4 ml/h for 72 h. A basal period of variable duration (8 to 12 h), during which hormone secretion was stable, was followed by steadily increasing secretion rates. Changes in cell number were not sufficient to acount for increased jormone secretion rates: a) there was no significant change in cell count after 72 h (0.97±0.03×10⁷;n=18); b) mean cell column DNA content increased 25.5% above the base value, whereas GH secretion rose 385% and PRL rose 178% (n=5). Observed differences in the duration of the basal secretion period, the basal secretory rate, and the magnitude of secretory rate increase were associated with several variables: a) variablility within a subline was a function of passage number: GH secretion decreased and PRL secretion increased with subculture number; b) cells with identical lot and freeze numbers, but received at different times, behaved differently; c) the presence of an antifungal agent (nystatin) altered hormone secretion reproducibly. Conclusions: a) rates of GH and PRL secretion rise spontaneously in perifusion without a proportional increase in GH₃ cell number; b) fluctuations in the rate of GH₃ cell secretion of GH and PRL are not entirely random but are determined by several definable variables. Supported by a grant to MES from the National Institutes of Health (AM33388) and in part by the Medical Research Service of the Veterans Administration.     

Identification of a putative egg-laying hormone in neural and ovarian tissues of the black tiger shrimp, Penaeus monodon, using immunocytochemistry

The existence of an egg-laying hormone (ELH) was identified for the first time in the black tiger shrimp, Penaeus monodon, by means of immunoenzyme and immunofluorescence techniques. This was achieved using a polyclonal antibody produced against expressed recombinant ELH of the female Australian blacklip abalone, Haliotis rubra. The shrimp ELH reactive material was found to be localised within female neurosecretory tissues and the secretory tissue of the antennal gland, but was not identified in the X-organ sinus gland within the eyestalk. It was also present in the ovary, where the amount of ELH present was observed to be greatest in the period prior to spawning. These findings implied that the induction of P. monodon spawning might be involved with humoral regulation relating to ELH expression.     

Impacts of the season and reproductive status on fecal reproductive and adrenocortical steroid metabolites in zoo Cuban crocodiles (Crocodylus rhombifer)

Conservation strategies for crocodilians often include captive breeding to create stable assurance populations. Evaluating adrenal and gonadal hormone patterns can provide animal managers with data to more effectively monitor animal welfare and reproductive status. This study evaluated the effects of season (breeding, nesting, or off), sex (male and female), and reproductive status of females (egg-laying/housed with a male or non-laying/housed solo) on concentrations of fecal glucocorticoid metabolite (FGM), fecal androgen metabolite (FAM), and fecal progestogen metabolite (FPM) in seven Cuban crocodiles, Crocodylus rhombifer, at the Smithsonian's National Zoological Park. Overall, seasonal changes in FGM and FPM concentrations were only observed in egg-laying females; FGM and FPM concentrations were both higher during the nesting season compared to the breeding and off seasons. Seasonal changes in FAM concentrations were only observed in males; males had higher FAM concentrations during the breeding and nesting seasons compared to the off season. Future studies investigating the use of fecal hormone metabolites in crocodilians are necessary to understand differences between individuals and species, to further elucidate the interactions between hormones and environmental factors, such as social housing, and to develop long-term datasets for the management of this species.     

Effects of starvation and hormones on DNA synthesis in silk gland cells of the silkworm,Bombyx mori

Silk gland cells of silkworm larvae undergo multiple cycles of endomitosis for the synthesis of silk proteins during the spinning phase. In this paper, we analyzed the endomitotic DNA synthesis of silk gland cells during larval development, and found that it was a periodic fluctuation, increasing during the vigorous feeding phase and being gradually inhibited in the next molting phase. That means it might be activated by a self‐regulating process after molting. The expression levels of cyclin E, cdt1 and pcna were consistent with these developmental changes. Moreover, we further examined whether these changes in endomitotic DNA synthesis resulted from feeding or hormonal stimulation. The results showed that DNA synthesis could be inhibited by starvation and re‐activated by re‐feeding, and therefore appears to be dependent on nutrition. DNA synthesis was suppressed by in vivo treatment with 20‐hydroxyecdysone (20E). However, there was no effect on DNA synthesis by in vitro 20E treatment or by either in vivo or in vitro juvenile hormone treatment. The levels of Akt and 4E‐BP phosphorylation in the silk glands were also reduced by starvation and in vivo treatment with 20E. These results indicate that the activation of endomitotic DNA synthesis during the intermolt stages is related to feeding and DNA synthesis is inhibited indirectly by 20E.     

Histophysiological studies on the corpus allatum during prepupal diapause in Monema flavescens (Lepidoptera)

The appearance of the corpus allatum, the central endocrine gland of diapause, was examined histologically in the slug moth prepupae, Monema flavescens (Lepidoptera) Before beginning of diapause, the secretory cells of the corpus allatum increase in size with the enrichment of the cytoplasm in quantity and in number of organelles, while RNA synthesis also starts. As diapause progresses, the secretory cells have many large unstained vacuoles in the cytoplasm, which were demonstrated to contain some substances of lipoidal nature. It is not clear that the substance is juvenile hormone itself or a material related to juvenile hormone. Agranular ER is the most characteristic organelle connected with mitochondria and situated around the vacuoles during diapause. Electron micrographs show that agranular ER and mitochondria have an essential role for the production of juvenile hormone. The function of the corpus allatum during diapause and the relationship between juvenile hormone and diapause are discussed.     

Hormonal control of egg-laying in the marine pulmonate,Onchidium verruculatum

Extirpation of the pleurovisceral ganglia (PV) blocked egg-laying whereas reimplantation of PV or injection of a PV homogenate into PV-less animals restored the egg-laying capacity of the marine pulomonate, O. verruculatum. Injection of PV homogenate induced egg-laying in intact, mature individuals within 15–45 minutes. Boiling of the homogenate for 15 minutes did not abolish its egg-laying efficacy. However, after incubation with trypsin, a PV homogenate lost its potency. Homogenates of PV from colchicinized animals could not induce egg-laying in intact Onchidium. Similarly, colchicinized animals did not respond to a PV ectract from intact Onchidium. It is hypothesized that egg-laying in O. verruculatum is under the control of a heat-stable, trypsin sensitive hormone secreted by the PV ganglia.     

Isolation of an egg-laying hormone-binding protein from the gonad of Aplysia californica and its localization in oocytes

A protein solubilized from a membrane preparation of the gonad of Aplysia californica has been isolated by affinity chromatography, using bag cell egg-laying hormone (ELH) as the bound ligand, and partially purified and characterized by gel electrophoresis. The protein has an apparent molecular weight of 52 kDa and consists of two disulfide-linked subunits of about 30 kDa each. The protein is glycosylated and has an acidic pI. Approximately 10–15 g of this protein can be isolated from a single ovotestis, representing less than 1% of the total protein in the gonad; but the protein could not be detected in buccal mass or body wall, tissues which do not have apparent response to ELH.Antibodies generated against this ELH-binding protein (ELHBP) were used to localize sites in the ovotestis which might contain this molecule and thus represent targets for egg-laying hormone. Immunocytochemical results indicate that the oocytes are a rich source of this protein, since their cytoplasm was the only detectable site of immunoreactivity.Whether this binding protein represents an egg-laying hormone receptor is uncertain, but its prevalence in oocytes suggests that ELH plays a signaling role on these gametes.Abbreviations ConA convalin A - DAB diaminobenzidine - ELH egg-laying hormone - ELHBP ELH-binding protein - IEF isoelectric focusing - IGFR insulin-like growth factor - IgG immunoglobulin - NGS normal goat serum - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate     

Morphometric evaluation of subcellular changes induced by in vivo TRH treatment in the pituitary gland of Rana perezi: Effects on prolactin and thyrotropic cells

Summary The effects of synthetic thyrotropin-releasing hormone on pituitary prolactin and thyrotropic cells were investigated in adult male Rana perezi (formerly Rana ridibunda) frogs. Animals were given daily injections of synthetic thyrotropin-releasing hormone into the dorsal lymph sac. Prolactin and thyrotropic cells were identified by the colloidal-gold method, using anti-human prolactin and anti-human--thyrotropin hormone as primary antisera. The stereological parameters of the rough endoplasmic reticulum, Golgi complex, and secretory granules of prolactin and thyrotropic cells were evaluated by ultrastructural morphometry (point-counting method). Thyrotropin-releasing hormone caused cytological changes in both cell-types which were consistent with increased synthesis and release of both prolactin and thryrotropin. These changes were still significant after 48 h treatment in the case of thyrotropic cells, while in prolactin cells the thyrotropin-releasing hormone increased the number of secretory granules. After 6 days, the cells resembled essentially those used as controls. These results indicate that thyrotropin-releasing hormone stimulates the synthesis and release of prolactin and thyrotropin, and that the response of each cell type to this hypothalamic stimulus follows a different time-course.This work has been supported by grants no. 2184-83 and PB 86-0095 from the Comisión Interministerial para la Ciencia y Tecnología, Spain     

Neuronal input contributes to sequence of Aplysia egg laying behaviors

Egg laying in Aplysia is controlled by the bag cell neuroendocrine system, which releases multiple peptides during a long-lasting electrical discharge. Following the discharge, a fixed sequence of head and neck movements is performed in which two phases can be distinguished: an appetitive or preparatory phase, in which the substrate is prepared, and a consummatory phase, when the egg string is deposited. During egg laying, feeding responses are suppressed. In this study, Aplysia fasciata was used. When the movement of the egg string through the genital groove was prevented by ligation, lesions of the nerve innervating the genital pore completely abolished the consummatory egg-laying behaviors. This shows that a nervous connection between the genital pore area and the central nervous system is important for the consummatory egg-laying behaviors.We found that suppression of feeding responses to seaweed occurred only during the consummatory phase of egg laying in controls, but animals with ligated genital grooves continued to show normal responses to food. It is hypothesized that a neuronal feedback, possibly together with the bag cell peptides, is critical for the temporal organization of egg-laying behavior in Aplysia.Abbreviations CNS central nervous system - ELH egg laying hormone     

Anatomy and ultrastructure of the floral nectary of Tontelea micrantha (Celastraceae: Salacioideae)

Among several native species of the Brazilian cerrado, a shrub, Tontelea micrantha, is exploited by traditional communities for the valuable oil extracted from its seeds, which has anti‐inflammatory properties. There have been no studies on the anatomy of its flower, and so the aim of this study is to describe the anatomy and ultrastructure of its floral nectary. Flower buds and flowers in anthesis were collected, fixed and processed for light and electron microscopy. The discoid floral nectary is composed of epidermis and a secretory parenchyma. Secretory cells are rich in plastids with starch grains and mitochondria. The nectar, sucrose dominant, is just sufficient to form a thin film on the nectary. The secretory cells show starch and oil droplets; however, during nectar production there is no evidence of hydrolysis of starch and some lipid reserves remain unchanged. Our results suggest a reduction in the amount of oil in the secretory cells during the secretory phase but this does not appear to imply a release of oil as a nectar component. In addition to maintaining part of the reserves, the lower frequency of organelles involved in nectar synthesis reinforces the hypothesis that phloem sap is the origin of nectar sugars. The tiny nectar film, released through modified stomata, is attractive to small insects such as flies. Considering the importance and intensity of use of T. micrantha in the Brazilian cerrado, we think that these data about its floral nectary can help to better explain its reproductive biology with positive impacts on its management and conservation.     

Melatonin in the seasonal response of the aphid Acyrthosiphon pisum

Aphids display life cycles largely determined by the photoperiod.During the warm long-day seasons.most aphid species reproduce by viviparous parthenogenesis.The shortening of the photoperiod in autumn induces a switch to sexual reproduction.Males and sexual females mate to produce overwintering resistant eggs.In addition to this full life cycle(holocycle),there are anholocyelic lineages that do not respond to changes in photoperiod and reproduce continuously by parthenogenesis.The molecular or hormonal events that trigger the scasonal response(i.c,induction of the sexual phenotypes)are still unknown.Although circadian synthesis of melatonin is known to play a key role in vertebrate photoperiodism,the involvement of the circadian clock and/or of the hor-mone melatonin in insect seasonal responses is not so well established.Here we show that melatonin levels in the aphid Acyrthosiphon pisum are significantly higher in holocyclice aphids reared under short days than under long days,while no differences were found between anholoeyelic aphids under the same conditions.We also found that melatonin is localized in the aphid suboesophageal ganglion(SOG)and in the thoracic ganglionic mass(TGM).In analogy to vertcbrates,insect-type arylalkxylamine N-acetyltransferases(i-AANATs)are thought to play a key role in melatonin synthesis.We measured the expression of four I-AANAT genes identified in A.pisum and localized two of them in situ in the insect central nervous systems(CNS).Levels of expression of these genes were compatible with the quantities of melatonin observed.Moreover,like melatonin,expression of these genes was found in the SOG and the TGM.     

Atrial gland induction of the egg-laying response inAplysia californica

Summary The atrial gland of the large hermaphroditic duct of the reproductive tract ofAplysia californica contains a substance that will cause egg laying when injected into sexually mature recipient animals. Preliminary chemical characterization of the atrial gland factor indicates that it is quite similar to the egg-laying hormone synthesized and secreted by the bag cell neuroendocrine organs of the central nervous system. In view of the apparent identity of its effects with those of the egg-laying hormone on presumed neural and reproductive system targets, it is possible that the atrial gland factor plays an hitherto unsuspected role in reproductive regulation. Moreover, the molecular similarity between the atrial gland factor and the egg-laying hormone suggests the possibility that the same humorally active species may be elaborated by two distinct tissue types.This work was supported by grants from the Medical Research Foundation of Oregon and USPHS (NS 11149). C. McC. was recipient of a Zlinkoff Research Fellowship. Dr. R. Beeman kindly confirmed our identification of the location and general morphology of the atrial gland.     

A new species of Sensoriaphis Cottier, 1953 (Homoptera: Aphididae) from New South Wales

The fundatrices, alatae viviparae, males, oviparae, eggs and egg-laying procedure of a new species of Sensoriaphis from Nothofagus moorei are described. An account is given of the unusual composition and seasonal distribution of its morphs. The eggs and egg-laying procedure of Neophyl-laphis brimblecombei Carver are also described.