Egg-laying Hormone Research Articles

Structural and functional characterization of an egg-laying hormone signaling system in a lophotrochozoan – The pacific oyster (Crassostrea gigas)

The egg-laying hormones (ELHs) of gastropod mollusks were characterized more than forty years ago. Yet, they have remained little explored in other mollusks. To gain insights into the functionality of the ELH signaling system in a bivalve mollusk – the oyster Crassostrea gigas, this study investigates the processing of its ELH precursor (Cragi-ELH) by mass spectrometry. Some of the ELH mature peptides identified in this study were subsequently investigated by nuclear magnetic resonance and shown to adopt an extended alpha-helix structure in a micellar medium mimicking the plasma membrane. To further characterize the ELH signaling system in C. gigas, a G protein-coupled receptor phylogenetically related to ecdysozoan diuretic hormone DH44 and corticotropin-releasing hormone (CRH) receptors named Cragi-ELHR was also characterized functionally and shown to be specifically activated by the two predicted mature ELH peptides and their N-terminal fragments. Both Cragi-ELH and Cragi-ELHR encoding genes were mostly expressed in the visceral ganglia (VG). Cragi-ELH expression was significantly increased in the VG of both fully mature male and female oysters at the spawning stage. When the oysters were submitted to a nutritional or hyposaline stress, no change in the expression of the ligand or receptor genes was recorded, except for Cragi-ELHR only during a mild acclimation episode to brackish water. These results suggest a role of Cragi-ELH signaling in the regulation of reproduction but not in mediating the stress response in our experimental conditions.

Hydrogen peroxide and phosphoinositide metabolites synergistically regulate a cation current to influence neuroendocrine cell bursting.

In various neurons, including neuroendocrine cells, non-selective cation channels elicit plateau potentials and persistent firing. Reproduction in the marine snail Aplysia californica is initiated when the neuroendocrine bag cell neurons undergo an afterdischarge, that is, a prolonged period of enhanced excitability and spiking during which egg-laying hormone is released into the blood. The afterdischarge is associated with both the production of hydrogen peroxide (H2 O2 ) and activation of phospholipase C (PLC), which hydrolyses phosphatidylinositol-4,5-bisphosphate into diacylglycerol (DAG) and inositol trisphosphate (IP3 ). We previously demonstrated that H2 O2 gates a voltage-dependent cation current and evokes spiking in bag cell neurons. The present study tests if DAG and IP3 impact the H2 O2 -induced current and excitability. In whole-cell voltage-clamped cultured bag cell neurons, bath-application of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a DAG analogue, enhanced the H2 O2 -induced current, which was amplified by the inclusion of IP3 in the pipette. A similar outcome was produced by the PLC activator, N-(3-trifluoromethylphenyl)-2,4,6-trimethylbenzenesulfonamide. In current-clamp, OAG or OAG plus IP3 , elevated the frequency of H2 O2 -induced bursting. PKC is also triggered during the afterdischarge; when PKC was stimulated with phorbol 12-myristate 13-acetate, it caused a voltage-dependent inward current with a reversal potential similar to the H2 O2 -induced current. Furthermore, PKC activation followed by H2 O2 reduced the onset latency and increased the duration of action potential firing. Finally, inhibiting nicotinamide adenine dinucleotide phosphate oxidase with 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine diminished evoked bursting in isolated bag cell neuron clusters. These results suggest that reactive oxygen species and phosphoinostide metabolites may synergize and contribute to reproductive behaviour by promoting neuroendocrine cell firing. KEY POINTS: Aplysia bag cell neurons secrete reproductive hormone during a lengthy burst of action potentials, known as the afterdischarge. During the afterdischarge, phospholipase C (PLC) hydrolyses phosphatidylinositol-4,5-bisphosphate into diacylglycerol (DAG) and inositol trisphosphate (IP3 ). Subsequent activation of protein kinase C (PKC) leads to H2 O2 production. H2 O2 evokes a voltage-dependent inward current and action potential firing. Both a DAG analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG), and IP3 enhance the H2 O2 -induced current, which is mimicked by the PLC activator, N-(3-trifluoromethylphenyl)-2,4,6-trimethylbenzenesulfonamide. The frequency of H2 O2 -evoked afterdischarge-like bursting is augmented by OAG or OAG plus IP3 . Stimulating PKC with phorbol 12-myristate 13-acetate shortens the latency and increases the duration of H2 O2 -induced bursts. The nicotinamide adenine dinucleotide phosphate oxidase inhibitor, 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine, attenuates burst firing in bag cell neuron clusters.

Hydrogen Peroxide Gates a Voltage-Dependent Cation Current in Aplysia Neuroendocrine Cells.

Nonselective cation channels promote persistent spiking in many neurons from a diversity of animals. In the hermaphroditic marine-snail, Aplysia californica, synaptic input to the neuroendocrine bag cell neurons triggers various cation channels, causing an ∼30 min afterdischarge of action potentials and the secretion of egg-laying hormone. During the afterdischarge, protein kinase C is also activated, which in turn elevates hydrogen peroxide (H2O2), likely by stimulating nicotinamide adenine dinucleotide phosphate oxidase. The present study investigated whether H2O2 regulates cation channels to drive the afterdischarge. In single, cultured bag cell neurons, H2O2 elicited a prolonged, concentration- and voltage-dependent inward current, associated with an increase in membrane conductance and a reversal potential of ∼+30 mV. Compared with normal saline, the presence of Ca2+-free, Na+-free, or Na+/Ca2+-free extracellular saline, lowered the current amplitude and left-shifted the reversal potential, consistent with a nonselective cationic conductance. Preventing H2O2 reduction with the glutathione peroxidase inhibitor, mercaptosuccinate, enhanced the H2O2-induced current, while boosting glutathione production with its precursor, N-acetylcysteine, or adding the reducing agent, dithiothreitol, lessened the response. Moreover, the current generated by the alkylating agent, N-ethylmaleimide, occluded the effect of H2O2 The H2O2-induced current was inhibited by tetrodotoxin as well as the cation channel blockers, 9-phenanthrol and clotrimazole. In current-clamp, H2O2 stimulated burst firing, but this was attenuated or prevented altogether by the channel blockers. Finally, H2O2 evoked an afterdischarge from whole bag cell neuron clusters recorded ex vivo by sharp-electrode. H2O2 may regulate a cation channel to influence long-term changes in activity and ultimately reproduction.SIGNIFICANCE STATEMENT Hydrogen peroxide (H2O2) is often studied in a pathological context, such as ischemia or inflammation. However, H2O2 also physiologically modulates synaptic transmission and gates certain transient receptor potential channels. That stated, the effect of H2O2 on neuronal excitability remains less well defined. Here, we examine how H2O2 influences Aplysia bag cell neurons, which elicit ovulation by releasing hormones during an afterdischarge. These neuroendocrine cells are uniquely identifiable and amenable to recording as individual cultured neurons or a cluster from the nervous system. In both culture and the cluster, H2O2 evokes prolonged, afterdischarge-like bursting by gating a nonselective voltage-dependent cationic current. Thus, H2O2, which is generated in response to afterdischarge-associated second messengers, may prompt the firing necessary for hormone secretion and procreation.

Greenlip Abalone (Haliotis laevigata) Genome and Protein Analysis Provides Insights into Maturation and Spawning.

Wild abalone (Family Haliotidae) populations have been severely affected by commercial fishing, poaching, anthropogenic pollution, environment and climate changes. These issues have stimulated an increase in aquaculture production; however production growth has been slow due to a lack of genetic knowledge and resources. We have sequenced a draft genome for the commercially important temperate Australian ‘greenlip’ abalone (Haliotis laevigata, Donovan 1808) and generated 11 tissue transcriptomes from a female adult abalone. Phylogenetic analysis of the greenlip abalone with reference to the Pacific abalone (Haliotis discus hannai) indicates that these abalone species diverged approximately 71 million years ago. This study presents an in-depth analysis into the features of reproductive dysfunction, where we provide the putative biochemical messenger components (neuropeptides) that may regulate reproduction including gonad maturation and spawning. Indeed, we isolate the egg-laying hormone neuropeptide and under trial conditions induce spawning at 80% efficiency. Altogether, we provide a solid platform for further studies aimed at stimulating advances in abalone aquaculture production. The H. laevigata genome and resources are made available to the public on the abalone ‘omics website, http://abalonedb.org.

Extensive conservation of the proneuropeptide and peptide prohormone complement in mollusks

As one of the most diverse groups of invertebrate animals, mollusks represent powerful models for neurobiological and developmental studies. Neuropeptides and peptide hormones are a heterogeneous class of signalling molecules involved in chemical communication between neurons and in neuroendocrine regulation. Here we present a fine-grained view of the molluscan neuropeptide and peptide hormone toolkit. Our results expand the distribution of several peptide families (e.g., prokineticin, insulin-related peptides, prohormone-4, LFRFamide) within Lophotrochozoa and provide evidence for an early origin of others (e.g., GNXQN/prohormone-2, neuroparsin). We identified a new peptide family broadly distributed among conchiferan mollusks, the PXRX family. We found the Wnt antagonist dickkopf1/2/4 ortholog in lophotrochozoans and nematodes and reveal that the egg-laying hormone family is a DH44 homolog restricted to gastropods. Our data demonstrate that numerous peptides evolved much earlier than previously assumed and that key signalling elements are extensively conserved among extant mollusks.

Existence of an egg-laying hormone-like peptide in male reproductive system of the giant freshwater prawn, Macrobrachium rosenbergii

The giant freshwater prawn, Macrobrachium rosenbergii, is an important aquaculture species. A better understanding of the molecular components of reproduction in this species would help to advance the prawn production. In the present study, we demonstrated the presence of an egg laying hormone (ELH)-like peptide in the male reproductive system. First, an antibody to the abalone (a)ELH was generated, and by Western blot it was shown to specifically bound to a protein from the male M. rosenbergii reproductive tissues with a similar size to molluscan ELH. This aELH-like peptide was localized in spermatogonia in the testes of all three male morphotypes: blue claw, orange claw and small males. Moreover, the aELH-like peptide was detected in the epithelium of the spermatic duct and its associated smooth muscle cell layers and on the outer surface of spermatozoa. As well, the aELH-like peptide was detected in the spermatophore located in the female thelycum at 4–6 h post-mating, indicating that it was transferred to the female during copulation. Taken together, we suggest that this aELH-like peptide could be as a male inducing factor that helped to accelerate female spawning. Liquid chromatography of crude extracts and immunoblot analysis suggested that the aELH-like peptide could be further purified for ultimate characterization.

Distribution of abalone egg-laying hormone-like peptide in the central nervous system and reproductive tract of the male mud crab, Scylla olivacea

The mud crab, Scylla olivacea, is a high value economic marine animal in Thailand. However, collection of these crabs from natural habitat for local consumption and export has caused rapid population decline. Hence, aquaculture of this species is required and to this measure understanding of endocrine control of their reproduction must be understood. Egg laying hormone (ELH) is a neuropeptide synthesized by the bag cells (neurons) in the abdominal ganglia of Aplysia gastropods. It plays a critical role in controlling egg production and laying in gastropods, and its possible homolog (ELH-like peptide) was reported in the neural and ovarian tissues of prawns and recently in female reproductive tract of the blue swimming crab, Portunus pelagicus. In this study, we have studied the histology of the male reproductive tract in Scylla olivacea which are comprised of anterior testis, posterior testis, early proximal spermatic duct (ePSD), proximal spermatic duct (PSD), middle spermatic duct (MSD) and distal spermatic duct (DSD), by immunohistochemistry, detected an abalone ELH- immunoreactivity (aELH-ir) in epithelium of ducts in posterior testis and epithelium of all parts of spermatic duct. Furthermore, we could detect aELH-ir in neurons of cluster 9, 11, olfactory neuropil (ON) in the brain and in the small neurons located between the third and the fourth thoracic neuropils (T3-T4) and between the fourth and the fifth thoracic neuropils (T4-T5) of thoracic ganglia. Thus, the presence of aELH in male S. olivacea was designated the role of female egg laying behavior in the male mud crab.

A Closely Associated Phospholipase C Regulates Cation Channel Function through Phosphoinositide Hydrolysis.

In the hemaphroditic sea snail, Aplysia californica, reproduction is initiated when the bag cell neurons secrete egg-laying hormone during a protracted afterdischarge. A source of depolarization for the afterdischarge is a voltage-gated, nonselective cation channel, similar to transient receptor potential (TRP) channels. Once the afterdischarge is triggered, phospholipase C (PLC) is activated to hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol trisphosphate (IP3). We previously reported that a DAG analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG), activates a prominent, inward whole-cell cationic current that is enhanced by IP3 To examine the underlying mechanism, we investigated the effect of exogenous OAG and IP3, as well as PLC activation, on cation channel activity and voltage dependence in excised, inside-out patches from cultured bag cell neurons. OAG transiently elevated channel open probability (PO) when applied to excised patches; however, coapplication of IP3 prolonged the OAG-induced response. In patches exposed to OAG and IP3, channel voltage dependence was left-shifted; this was also observed with OAG, but not to the same extent. Introducing the PLC activator, m-3M3FBS, to patches increased channel PO, suggesting PLC may be physically linked to the channels. Accordingly, blocking PLC with U-73122 ablated the m-3M3FBS-induced elevation in PO Treatment with m-3M3FBS left-shifted cation channel voltage dependence to a greater extent than exogenous OAG and IP3 Finally, OAG and IP3 potentiated the stimulatory effect of PKC, which is also associated with the channel. Thus, the PLC-PKC signaling system is physically localized such that PIP2 breakdown products liberated during the afterdischarge modulate the cation channel and temporally influence neuronal activity.SIGNIFICANCE STATEMENT Using excised patches from Aplysia bag cell neurons, we present the first evidence of a nonselective cation channel physically associating with phospholipase C (PLC) at the single-channel level. PLC-mediated breakdown of phospholipids generates diacylglycerol and inositol trisphosphate, which activate the cation channel. This is mimicked by exogenous lipids; furthermore, these second messengers left-shift channel voltage dependence and enhance the response of the channel to protein kinase C. PLC-mediated lipid signaling controls single-channel currents to ensure depolarization is maintained for an extended period of firing, termed the afterdischarge, when the bag cell neurons secrete egg-laying hormone to trigger reproduction.

Tyrosine Phosphorylation Determines Afterdischarge Initiation by Regulating an Ionotropic Cholinergic Receptor

Changes to neuronal activity often involve a rapid and precise transition from low to high excitability. In the marine snail, Aplysia, the bag cell neurons control reproduction by undergoing an afterdischarge, which begins with synaptic input releasing acetylcholine to open an ionotropic cholinergic receptor. Gating of this receptor causes depolarization and a shift from silence to continuous action potential firing, leading to the neuroendocrine secretion of egg-laying hormone and ovulation. At the onset of the afterdischarge, there is a rise in intracellular Ca2+, followed by both protein kinase C (PKC) activation and tyrosine dephosphorylation. To determine whether these signals influence the acetylcholine ionotropic receptor, we examined the bag cell neuron cholinergic response both in culture and isolated clusters using whole-cell and/or sharp-electrode electrophysiology. The acetylcholine-induced current was not altered by increasing intracellular Ca2+ via voltage-gated Ca2+ channels, clamping intracellular Ca2+ with exogenous Ca2+ buffers, or activating PKC with phorbol esters. However, lowering phosphotyrosine levels by inhibiting tyrosine kinases both reduced the cholinergic current and prevented acetylcholine from triggering action potentials or afterdischarge-like bursts. In other systems, acetylcholine receptors are often modulated by multiple signals, but bag cell neurons appear to be more restrictive in this regard. Prior work finds that, as the afterdischarge proceeds, tyrosine dephosphorylation leads to biophysical alterations that promote persistent firing. Because this firing is subsequent to the cholinergic input, inhibiting the acetylcholine receptor may represent a means of properly orchestrating synaptically induced changes in excitability.

The presence of abalone egg-laying hormone-like peptide in the central nervous system and ovary of the Spotted Babylon, Babylonia areolata

Recently, the neuronal classification of the ivory shell Spotted Babylon, Babylonia areolata, was readily demonstrated. Regarding its importance as marine economic molluscan species, the attempt to understand the neuroendocrine regulation is necessary. This study firstly demonstrated the neurosecretory cells as well as the existence and distribution of the egg-laying hormone (ELH)-like peptide in the central nervous system (CNS) and ovary of the B. areolata. The neurosecretory cell was characterized by the cytoplasmic purple dot-like structure as stained by the Gomori’s paraldehyde fuchsin. Using the anti-abalone (a) ELH, we detected the aELH-like-peptide in neurons (Nr) and neurosecretory cells (Ns) of all ganglia including the cerebral, pleural, parietal, pedal and buccal ganglia. The aELH-like peptide was also present in the neuropil of each. It was noted that not all Ns presented the aELH-like peptide. In the ovary, the aELH-like peptide was slightly detected in early developing oocytes and strongly detected in late developing oocytes and follicular cells. This study firstly reported the evidence of ELH-like peptide in the CNS and ovary of the B. areolata. The molecular cloning as well as to investigate the function of ELH in this species is needed as it will be beneficial for future applications in aquaculture.

The presence of abalone egg-laying hormone-like peptide in the central nervous system and ovary of the blue swimming crab, Portunus pelagicus, and its effect on ovarian maturation

Egg-laying hormone (ELH) is a neuropeptide secreted from the neural ganglia of mollusks that can stimulate the egg-laying and associated behaviors. In the present study, using a polyclonal antibody against an abalone, H asinina, ELH (aELH) we could detect the aELH-like immunoreactivity (aELH-l-ir) in the central nervous system (CNS) and ovary of the blue swimming crab Portunus pelagicus. In protocerebrum, aELH-l-ir was detected in the small- and medium- sized neurons of cluster 6, anterior/posterior medial protocerebral neuropils, protocerebral bridge and central body. In deutocerebrum, the aELH-l-ir was observed in small-sized neurons of cluster 9, fibers between small-sized neurons of cluster 10, medium-sized neurons of cluster 11, olfactory neuropil and medial/lateral antenna I neuropils. In tritocerebrum, the medium- sized neurons of cluster 16, the tritocerebral commissure, and tegumental neuropil showed strong aELH-l-ir. In the ventral nerve cord, the aELH-l-ir was detected in the small- and medium-sized neurons of dorsolateral clusters of subesophageal ganglion. There was no aELH-l- ir in neurons of thoracic ganglion, but aELH-l-ir exhibited a dot-like structure in the subcapsular space around the thoracic neuropils. In abdominal ganglion, the aELH-l-ir was detected in some medium-sized neurons and abdominal neuropils. In the ovary, the aELH-l-ir was detected in the cytoplasm of oogonia, and all steps of developing oocytes. The effect of aELH on ovarian maturation in the crabs was examined by bioassay. When mature female crabs were injected with a dose of 500ng/g body weight per crab, the ovarian maturation was accelerated as indicated by the rising of gonado-somatic index and levels of vitellin in the ovarian tissue and hemolymph, which was confirmed by histological studies. The aELH also enhanced the proliferation of follicular cells as indicated by the increased number of BrdU-labeled nuclei. In addition, the aELH also significantly induced the 17β-estradiol and progesterone secretion in the cultured ovarian explant. In conclusion, this study demonstrated the existence of an aELH-liked-peptide in the CNS and ovary of the P. pelagicus, and it can induce the ovarian 17β-estradiol and progesterone production resulting in early ovarian maturation in this crab.

Neuropeptides encoded within a neural transcriptome of the giant triton snail Charonia tritonis, a Crown-of-Thorns Starfish predator

Neuropeptides represent a diverse class of signaling molecules originating from neural tissues. These chemical modulators orchestrate complex physiological events including those associated with growth and reproduction. De novo transcriptome sequencing of a cerebral ganglion library of the endangered giant triton snail (Charonia tritonis) was undertaken in an effort to identify key neuropeptides that control or influence its physiology. The giant triton snail is considered a primary predator of the corallivore Acanthaster planci (Crown-of-Thorns Starfish) that is responsible for a significant loss in coral cover on reefs in the Indo-Pacific. The transcriptome library was assembled into contigs, and then bioinformatic analysis was used to identify a repertoire of 38 giant triton snail neuropeptide precursor genes, and various isoforms, that encode conserved molluscan neuropeptides. C. tritonis neuropeptides show overall precursor organisation consistent with those of other molluscs. These include those neuropeptides associated with mollusc reproduction such as the APGWamide, buccalin, conopressin, gonadotropin-releasing hormone (GnRH), NKY and egg-laying hormone. These data provide a foundation for further studies targeted towards the functional characterisation of neuropeptides to further understand aspects of the biology of the giant triton snail, such as elucidating its reproductive neuroendocrine pathway to allow the development of knowledge based captive breeding programs.

Characterisation of Reproduction-Associated Genes and Peptides in the Pest Land Snail, Theba pisana.

Increased understanding of the molecular components involved in reproduction may assist in understanding the evolutionary adaptations used by animals, including hermaphrodites, to produce offspring and retain a continuation of their lineage. In this study, we focus on the Mediterranean snail, Theba pisana, a hermaphroditic land snail that has become a highly invasive pest species within agricultural areas throughout the world. Our analysis of T. pisana CNS tissue has revealed gene transcripts encoding molluscan reproduction-associated proteins including APGWamide, gonadotropin-releasing hormone (GnRH) and an egg-laying hormone (ELH). ELH isoform 1 (ELH1) is known to be a potent reproductive peptide hormone involved in ovulation and egg-laying in some aquatic molluscs. Two other non-CNS ELH isoforms were also present in T. pisana (Tpi-ELH2 and Tpi-ELH3) within the snail dart sac and mucous glands. Bioactivity of a synthetic ELH1 on sexually mature T. pisana was confirmed through bioassay, with snails showing ELH1-induced egg-laying behaviours, including soil burrowing and oviposition. In summary, this study presents a detailed molecular analysis of reproductive neuropeptide genes in a land snail and provides a foundation for understanding ELH function.

Diacylglycerol-mediated regulation of Aplysia bag cell neuron excitability requires protein kinase C.

In Aplysia, reproduction is initiated by the bag cell neurons and a prolonged period of enhanced excitability known as the afterdischarge. Phosphoinositide turnover is upregulated during the afterdischarge resulting in the hydrolysis of phosphatidylinositol-4,5-bisphosphate by phospholipaseC (PLC) and the release of diacylglycerol (DAG) and inositol trisphosphate (IP3 ). In whole-cell voltage-clamped cultured bag cell neurons, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a synthetic DAG analogue, activates a dose-dependent, transient, inward current (IOAG ) that is enhanced by IP3 , mimicked by PLC activation and dependent on basal protein kinaseC (PKC) activity. OAG depolarizes bag cell neurons and triggers action potential firing in culture, and prolongs electrically stimulated afterdischarges in intact bag cell neuron clusters ex vivo. Although PKC alone cannot activate the current, it is required for IOAG ; this is the first description of required obligate PKC activity working in concert with PLC, DAG and IP3 to maintain the depolarization required for prolonged excitability in Aplysia reproduction. Following synaptic input, the bag cell neurons of Aplysia undergo a long-term afterdischarge of action potentials to secrete egg-laying hormone and initiate reproduction. Early in the afterdischarge, phospholipaseC (PLC) hydrolyses phosphatidylinositol-4,5-bisphosphate into inositol trisphosphate (IP3 ) and diacylglycerol (DAG). In Aplysia, little is known about the action of DAG, or any interaction with IP3 ; thus, we examined the effects of a synthetic DAG analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG), on whole-cell voltage-clamped cultured bag cell neurons. OAG induced a large, prolonged, Ca(2+) -permeable, concentration-dependent inward current (IOAG ) that reversed at ∼-20mV and was enhanced by intracellular IP3 . A similar current was evoked by either another DAG analogue, 1,2-dioctanoyl-sn-glycerol (DOG), or activating PLC with N-(3-trifluoromethylphenyl)-2,4,6-trimethylbenzenesulfonamide (m-3M3FBS). IOAG was reduced by the general cation channel blockers Gd(3+) or flufenamic acid. Work in other systems indicated that OAG activates channels independently of protein kinaseC (PKC); however, we found pretreating bag cell neurons with any of the PKC inhibitors bisindolylmaleimide, sphinganine, or H7, attenuated IOAG . However, stimulating PKC with phorbol 12-myristate 13-acetate (PMA) did not evoke current or enhance IOAG ; moreover, unlike PMA, OAG failed to trigger PKC, as confirmed by an independent bioassay. Finally, OAG or m-3M3FBS depolarized cultured neurons, and while OAG did not provoke afterdischarges from bag cell neurons in the nervous system, it did double the duration of synaptically elicited afterdischarges. To our knowledge, this is the first report of obligate PKC activity for IOAG gating. An interaction between phosphoinositol metabolites and PKC could control the cation channel to influence afterdischarge duration.

Ca2+-induced uncoupling of Aplysia bag cell neurons.

Electrical transmission is a dynamically regulated form of communication and key to synchronizing neuronal activity. The bag cell neurons of Aplysia are a group of electrically coupled neuroendocrine cells that initiate ovulation by secreting egg-laying hormone during a prolonged period of synchronous firing called the afterdischarge. Accompanying the afterdischarge is an increase in intracellular Ca2+ and the activation of protein kinase C (PKC). We used whole cell recording from paired cultured bag cell neurons to demonstrate that electrical coupling is regulated by both Ca2+ and PKC. Elevating Ca2+ with a train of voltage steps, mimicking the onset of the afterdischarge, decreased junctional current for up to 30 min. Inhibition was most effective when Ca2+ entry occurred in both neurons. Depletion of Ca2+ from the mitochondria, but not the endoplasmic reticulum, also attenuated the electrical synapse. Buffering Ca2+ with high intracellular EGTA or inhibiting calmodulin kinase prevented uncoupling. Furthermore, activating PKC produced a small but clear decrease in junctional current, while triggering both Ca2+ influx and PKC inhibited the electrical synapse to a greater extent than Ca2+ alone. Finally, the amplitude and time course of the postsynaptic electrotonic response were attenuated after Ca2+ influx. A mathematical model of electrically connected neurons showed that excessive coupling reduced recruitment of the cells to fire, whereas less coupling led to spiking of essentially all neurons. Thus a decrease in electrical synapses could promote the afterdischarge by ensuring prompt recovery of electrotonic potentials or making the neurons more responsive to current spreading through the network.

Voltage-gated Ca2+ influx and mitochondrial Ca2+ initiate secretion from Aplysia neuroendocrine cells

Neuroendocrine secretion often requires prolonged voltage-gated Ca2+ entry; however, the ability of Ca2+ from intracellular stores, such as endoplasmic reticulum or mitochondria, to elicit secretion is less clear. We examined this using the bag cell neurons, which trigger ovulation in Aplysia by releasing egg-laying hormone (ELH) peptide. Secretion from cultured bag cell neurons was observed as an increase in plasma membrane capacitance following Ca2+ influx evoked by a 5-Hz, 1-min train of depolarizing steps under voltage-clamp. The response was similar for step durations of ⩾50ms, but fell off sharply with shorter stimuli. The capacitance change was attenuated by replacing external Ca2+ with Ba2+, blocking Ca2+ channels, buffering intracellular Ca2+ with EGTA, disrupting synaptic protein recycling, or genetic knock-down of ELH. Regarding intracellular stores, liberating mitochondrial Ca2+ with the protonophore, carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP), brought about an EGTA-sensitive elevation of capacitance. Conversely, no change was observed to Ca2+ released from the endoplasmic reticulum or acidic stores. Prior exposure to FCCP lessened the train-induced capacitance increase, suggesting overlap in the pool of releasable vesicles. Employing GTP-γ-S to interfere with endocytosis delayed recovery (presumed membrane retrieval) of the capacitance change following FCCP, but not the train. Finally, secretion was correlated with reproductive behavior, in that neurons isolated from animals engaged in egg-laying presented a greater train-induced capacitance elevation vs quiescent animals. The bag cell neuron capacitance increase is consistent with peptide secretion requiring high Ca2+, either from influx or stores, and may reflect the all-or-none nature of reproduction.

Activity changes in jaw motor neurons induced by egg-laying hormone contribute to the feeding suppression during egg-laying behavior in Aplysia kurodai

Egg-laying behavior in Aplysia is accompanied by behavioral changes such as feeding suppression. We investigated the effects of the egg-laying hormone (ELH) on food intake, the activity patterns of jaw muscles, and the activity of buccal neurons (multi-action neuron [MA1] and jaw-closing motor neuron [JC2]), which are elements of the feeding neural circuits controlling jaw movements in Aplysia kurodai. Injection of ELH into the body cavity inhibited the intake of seaweed. After ELH application, the rhythmic activity of jaw muscles that was induced by preferred taste stimulation elicited fewer ingestion-like responses and increased the number of rejection-like responses. ELH applied to the buccal ganglia increased the firing activity of JC2 during spontaneous rhythmic responses and during the rhythmic feeding-like responses that were evoked by electrical stimulation of the esophageal nerves. In the 2 types of rhythmic responses, the Dn (normalized value of the delay time of JC2 firing onset) decreased after ELH application as compared with the control. Furthermore, ELH decreased the size of MA1-induced inhibitory postsynaptic currents in JC2. These results suggest that ELH changes the buccal motor program from ingestion to rejection on the basis of our previous results, and may contribute to a decrease in food intake during egg laying.

Stimulatory Effects of Egg-Laying Hormone and Gonadotropin-Releasing Hormone on Reproduction of the Tropical Abalone,Haliotis asininaLinnaeus

ABSTRACT Egg-laying hormone (ELH) is a neuropeptide hormone that stimulates ovulation of gastropods, including Aplysia californica and Lymnaea stagnalis. Other neuropeptides, gonadotropin releasing hormones (GnRHs), also play important roles in controlling reproduction in both vertebrates and invertebrates. In the current study, the effects of abalone ELH (aELH) and several GnRHs on somatic growth, sex differentiation, gonad maturation, and spawning of Haliotis asinina were investigated in 3 experiments. In experiment 1, groups of 4-mo-old juveniles (11.8 ± 0.03 mm shell length (SL) and 0.33 ± 0.04 g body weight (BW)) were injected with aELH and GnRHs, including buserelin (mammalian GnRH analogue), octopus GnRH (octGnRH), and tunicate GnRH-I (tGnRH-I), at doses of 20 ng/g BW and 200 ng/g BW. The aELH induced early sex differentiation with a bias toward females, but with normal somatic growth, whereas the different isoforms of GnRH had no effect on sexual differentiation or somatic growth. In experiment 2,...

Regulatory actions of neuropeptides and peptide hormones on the reproduction of molluscsThe present review is one of a series of occasional review articles that have been invited by the Editors and will feature the broad range of disciplines and expertise represented in our Editorial Advisory Board.

Reproductive success of individual animals is essential for the survival of any species. Molluscs have adapted to a wide variety of environments (freshwater, brackish water, seawater, and terrestrial habits) and have evolved unique tactics for reproduction. Both of these features attract the academic interests of scientists. Because neuropeptides and peptide hormones play critical roles in neural and neurohormonal regulation of physiological functions and behaviors in this animal group, the regulatory actions of these messengers in reproduction have been extensively investigated. In this review, we will briefly summarize how peptidergic messengers are involved in various aspects of reproduction, using some peptides such as egg-laying hormone, caudo-dorsal cell hormone, APGWamide, and gonadotropin-releasing hormone as typical examples.

Conservation of the egg-laying hormone neuropeptide and attractin pheromone in the spotted sea hare, Aplysia dactylomela

In the marine opisthobranch mollusc, Aplysia, secreted peptides and proteins play an essential role in egg laying and mate attraction. Aplysia californica egg laying is initiated by secretion of the egg-laying hormone (ELH) peptide while mate attraction is made possible by protein pheromones, such as attractin, released into the surrounding seawater with the egg cordon. In this study, we investigated the existence of similar egg-laying hormone and attractin products in the spotted sea hare, Aplysia dactylomela, a species that is widely distributed in almost all tropical and temperate oceans, including Australia's Great Barrier Reef. Immunological analysis revealed that an ELH-like transmitter is present within bag cell somata and processes of the abdominal ganglion. A molecular genetic approach found that the ELH precursor mRNA is synthesized in the abdominal ganglia and encodes a 36-residue peptide (dELH) that is cleaved from the prohormone prior to secretion. It is most closely related to A. californica and A. brasiliana ELH (91.7% identical). We also found that A. dactylomela synthesize an attractin pheromone in the albumen gland that is released during egg laying. The gene encodes a 58-residue mature protein that is 74.9% similar to A. californica attractin. We demonstrate that an increase in seawater temperature can disrupt attractins higher order interactions, such as those with the pheromone temptin, and accelerates attractin degradation. Together, these findings further expands our understanding of pheromone intermolecular interactions and presents an opportunity for further study of how increases in sea water temperature may affect this important marine communication system.