Egg Cordons Research Articles

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.

Characterization of Aplysia Alb-1, a candidate water-borne protein pheromone released during egg laying

Pheromones are known to be important to the innate behavior of marine animals. Attraction in Aplysia involves the long-distance water-borne protein pheromones attractin, enticin, temptin and seductin, which are released from the albumen gland during egg laying. Other pheromones are predicted to act in concert with these pheromones, but their identities are unknown. To identify additional pheromone candidates, we employed differential library screening of an albumen gland cDNA library, RT-PCR, recombinant protein expression, rhinophore contraction bioassays and immunocytochemistry. Alb-1 is expressed in the Aplysia californica albumen gland and encodes a novel protein that does not share significant sequence identity with any proteins in the database. RT-PCR analysis detected Alb-1 transcripts in the albumen gland, exocrine atrial gland and ovotestis. The Alb-1 precursor has a signal peptide sequence followed by a predicted 101-residue protein sequence containing eight cysteine residues. Recombinant protein expression, RP-HPLC, microsequence analysis and MALDI mass spectrometry analyses demonstrated that mature recombinant Alb-1 was processed at a paired basic residue site to generate an N-terminal and C-terminal protein fragment; this was consistent with immunoblot observations on purified albumen gland extracts. In rhinophore contraction (twitch) bioassays, the recombinant N-terminal protein induced rhinophore contractions whereas the C-terminal protein did not. An antibody generated to the N-terminal protein was used for immunocytochemical and immunoblot analyses and demonstrated that this protein is present in albumen gland secretory cells, egg cordons and egg eluates. Overall, the data suggest that Alb-1 may be processed in the albumen gland and that the Alb-1 1–56 protein released during egg laying may serve a pheromonal function in concert with attractin, enticin, temptin and seductin.

Aplysia temptin − the ‘glue’ in the water-borne attractin pheromone complex

Temptin, a component of the complex of water-borne protein pheromones that stimulate attraction and mating behavior in the marine mollusk Aplysia, has sequence homology to the epidermal growth factor (EGF)-like domains of higher organisms that mediate protein-cell surface contact during fertilization and blood coagulation. In this work, recombinant temptin for structural and functional studies was produced in Escherichia coli using a cold shock promoter and purified by RP-HPLC. CD spectra confirmed a predominantly beta-sheet structure. Two disulfide bonds were determined via limited proteolysis and MS. One internal disulfide (Cys57-Cys77) was predicted from initial alignments with class I EGF-like domains; the second, between Cys18 and Cys103, could protect temptin against proteolysis in seawater and stabilize its interacting surface. A three-dimensional model of temptin was prepared with our MPACK suite, based on the Ca(2+)-binding, EGF-like domain of the extracellular matrix protein fibrillin. Two temptin residues, Trp52 and Trp79, which align with cysteine residues conserved in fibrillins, lie adjacent to and could stabilize the disulfide bonds and a proposed metal-binding loop. The water-borne pheromone attractin in egg cordon eluates is complexed with other proteins. Docking results with our model and the NMR structure of attractin suggest that one face of temptin interacts with the pheromone, perhaps controlling its access to the cellular receptors. Gel shifts confirmed that temptin complexes with wild-type attractin. These results indicate that temptin, analogous to the role of fibrillin in controlling transforming growth factor-beta concentration, modulates pheromone signaling by direct binding to attractin.

Aplysia capsulin is localized to egg capsules and egg cordon sheaths and shares sequence homology with Drosophila dec-1 gene products

Differential library screening of an albumen gland cDNA library, Western blot analysis, protein expression, immunolocalization studies, comparative genomics, and secretion assays identified a major Aplysia californica albumen gland protein (‘capsulin’) that is localized to egg capsules and to the sheaths of the egg cordon. Capsulin shared sequence homology with eggshell proteins encoded by the Drosophila dec-1 gene. The 1790-amino acid A. californica precursor contains 17 repeat sequences that are flanked by basic residue processing sites. The numerous proteolytic processing sites may facilitate the breakdown of capsulin prior to when veliger larvae break out of egg capsules as free-swimming larvae. An Aplysia brasiliana capsulin repeat sequence was 97% identical to its A. californica homolog. Capsulin fragments were not detected in the eluates of egg cordons, suggesting that capsulin is not a candidate water-borne pheromone precursor.

Aplysia seductin is a water-borne protein pheromone that acts in concert with attractin to stimulate mate attraction

Mate attraction in Aplysia involves the long-distance water-borne protein pheromones attractin, enticin, and temptin which are released during egg-laying. Other water-borne pheromones are predicted to act in concert with attractin, enticin, and temptin, but their identities were unknown. We recently identified a highly expressed Aplysia californica albumen gland gene ( Alb-23) that encoded a novel protein by differential library screening of an albumen gland cDNA library. To determine whether Alb-23 (‘seductin’) was a water-borne pheromone, we employed Western blot analysis, purification and expression of albumen gland proteins, immunolocalization studies, pheromone secretion assays, comparative genomics, and behavioral bioassays. Immunoreactive seductin was detected in eluates of egg cordons, indicating that seductin was secreted onto the cordon during egg laying. Aplysia brasiliana seductin was 94% identical to its A. californica homolog. In T-maze attraction assays, the combination of attractin and seductin was significantly attractive to potential mates, whereas either protein alone was not. Data from this and previous studies support the hypothesis that seductin is a water-borne protein pheromone that acts in concert with attractin, enticin, and temptin to attract Aplysia to form and maintain mating aggregations.

Characterization of Aplysia Enticin and Temptin, Two Novel Water-borne Protein Pheromones That Act in Concert with Attractin to Stimulate Mate Attraction

Mate attraction in Aplysia involves a long-distance water-borne signal (attractin) that is released during egg laying. Other pheromones are predicted to be released during egg laying that act in concert with albumen gland attractin to stimulate attraction, but their identities are unknown. To identify other candidate water-borne pheromones, we employed differential library screening of an albumen gland cDNA library, Northern blot analysis, purification, characterization, cloning, and expression of albumen gland proteins, matrix-assisted laser desorption ionization mass spectrometry, pheromone secretion assays, behavioral bioassays, immunolocalization studies, and comparative genomics. Four genes, Alb-23, Alb-24, Alb-69, and Alb-172, were highly expressed in Aplysia californica albumen glands and encoded novel proteins. The products of the Alb-24 ("enticin") and Alb-172 ("temptin") precursors were soluble and highly abundant in albumen gland extracts, whereas Alb-23 and Alb-69 were membrane-associated proteins. A comparative analysis showed that the predicted Aplysia brasiliana enticin and temptin proteins were 90 and 91% identical, respectively, to their A. californica homologs. T-maze attraction bioassay studies have previously demonstrated that egg cordons alone are attractive to Aplysia but that attractin alone is not. In the present study, however, the combination of attractin, enticin, and temptin was found to be significantly attractive to potential mates and doubled the number of animals attracted to this stimulus compared with control animals. The combined data strongly suggest that enticin and temptin are novel candidate water-borne protein pheromones that act in concert with attractin to attract Aplysia to form and maintain egglaying and mating aggregations.

Peptide products of the atrial gland are not water-borne reproductive pheromones during egg laying in Aplysia

Mate attraction in Aplysia involves long-distance water-borne signaling via the secretion of the peptide pheromone attractin from the exocrine albumen gland during egg laying. Previous studies have shown that a second exocrine organ, the atrial gland, produces abundant egg-laying hormone (ELH) precursor-related peptides and mollusk-derived growth factor (MDGF), and crude extracts of the atrial gland are attractive in T-maze attraction assays. However, it is not known whether these peptides and proteins are secreted during egg laying. In this report, seawater eluates of freshly laid egg cordons were concentrated and fractionated by C18 RP-HPLC, and the resulting major peaks were examined by amino acid compositional analysis, microsequence analysis, and electrospray mass spectrometry. Concentrated egg cordon eluates were also examined by immunoblot analysis using anti-MDGF antisera as probe. The combined data demonstrated that the atrial gland of Aplysia californica does not secrete detectable levels of either ELH precursor-related peptides or MDGF during egg laying. Although the atrial gland is the last major exocrine organ to make contact with eggs before they are laid, the gland does not appear to secrete water-borne peptide pheromones during egg laying.

Aplysia Attractin: Biophysical Characterization and Modeling of a Water-Borne Pheromone

Attractin, a 58-residue protein secreted by the mollusk Aplysia californica, stimulates sexually mature animals to approach egg cordons. Attractin from five different Aplysia species are ∼40% identical in sequence. Recombinant attractin, expressed in insect cells and purified by reverse-phase high-performance liquid chromatography (RP-HPLC), is active in a bioassay using A. brasiliana; its circular dichroism (CD) spectrum indicates a predominantly α-helical structure. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) characterization of proteolytic fragments identified disulfide bonds between the six conserved cysteines (I–VI, II–V, III–IV, where the Roman numeral indicates the order of occurrence in the primary sequence). Attractin has no significant similarity to any other sequence in the database. The protozoan Euplotes pheromones were selected by fold recognition as possible templates. These diverse proteins have three α-helices, with six cysteine residues disulfide-bonded in a different pattern from attractin. Model structures with good stereochemical parameters were prepared using the EXDIS/DIAMOD/FANTOM program suite and constraints based on sequence alignments with the Euplotes templates and the attractin disulfide bonds. A potential receptor-binding site is suggested based on these data. Future structural characterization of attractin will be needed to confirm these models.

Attractin, a water-borne peptide pheromone in Aplysa

Summary The opisthobranch mollusk Aplysia is a simultaneous hermaphrodite that does not usually fertilize its own eggs. It is solitary during most of the year, but moves into breeding aggregations during the summer reproductive season. The aggregations contain both mating and egg-laying animals and are associated with masses of egg cordons. The egg cordons are a source of both contact and water-borne pheromones that attract animals to the area and induce or facilitate reproductive activity. Three characteristics of the Aplysia reproductive system make it ideal for the isolation and characterization of a water-borne peptide pheromone: (a) egg laying can be induced by injecting atrial gland extract into the hemocoel; (b) the resulting eggs are packaged into a long string or “cordon” which has a large surface area; and (c) the cordon is a source of water-borne pheromones. By using atrial gland extract to induce egg laying, it is possible to control the number of animals laying eggs, the timing and synchronization of egg laying, the general area in which the eggs are deposited and, to some extent, the purity of the seawater in which they are deposited. In terms of the resulting “cordon-derived” pheromones, this means that it is possible to control the timing of pheromone secretion, the amount of pheromone secreted, and the relative amount of contaminants in the seawater. This approach was used in the studies described in this paper to isolate and characterize a peptide that is a water-borne pheromonal attractant in Aplysia. To the best of our knowledge, it is the first water-borne peptide pheromone characterized in mollusks and in invertebrates. Some of these studies have been reported previously (Painter et al., 1998).

Characterization of Aplysia Attractin, the First Water-borne Peptide Pheromone in Invertebrates

Although animals in the genus Aplysia are solitary during most of the year, they form breeding aggregations during the reproductive season. The aggregations contain both mating and egg-laying animals and are associated with masses of egg cordons. The egg cordons are a source of pheromones that establish and maintain the aggregation, but none of the pheromonal factors have been chemically characterized. In these studies, specimens of Aplysia were induced to lay eggs, the egg cordons collected and eluted, and the eluates fractionated by C18 reversed-phase HPLC. Four peak fractions were bioassayed in a T-maze. All four increased the number of animals attracted to a nonlaying conspecific and were thus subjected to compositional and microsequence analysis. Each contained the same NH2-terminal peptide sequence. The full-length peptide ("attractin") was isolated from the albumen gland, a large exocrine organ that packages the eggs into a cordon. The complete 58-residue sequence was obtained, and it matched that predicted by an albumen gland cDNA. Mass spectrometry showed that attractin is 21 wt.% carbohydrate as the result of N-linked glycosylation. T-maze bioassays confirmed that the full-length peptide is attractive. Attractin is the first water-borne peptide pheromone characterized in molluscs, and the first in invertebrates.

Molecular cloning of a cDNA encoding a potential water-borne pheromonal attractant released during Aplysia egg laying

Recently deposited egg cordons are a source of water-borne pheromones that attract the marine mollusk Aplysia into breeding aggregations and coordinate male and female reproductive behavior within the aggregation. A potential pheromonal attractant has been isolated from egg cordon eluates and the peptide partially characterized [S.D. Painter, B. Clough, X. Fan, G.T. Nagle, Soc. Neurosci. Abstr., Vol. 22 (1996) 837]. Using this information, we have cloned an Aplysia albumen gland cDNA that encodes a precursor protein containing a single copy of the full-length peptide, and demonstrated that there are abundant levels of pheromone mRNA transcripts (0.8 and 2.5 kb) in the albumen gland. This is consistent with the reported function of the gland (i.e. packaging the eggs into a cordon for deposition), with behavioral studies showing that the albumen gland is a potential source of attractants, and more recent biochemical studies in which the full-length peptide has been isolated from the albumen gland. This is the first candidate peptide pheromone in mollusks and the first in invertebrates. The pheromonal regulatory system in Aplysia may provide a model system for examining the structural characteristics of peptide pheromones.

The rhinophores sense pheromones regulating multiple behaviors in Aplysia fasciata

Pheromones released during mating and egg laying in Aplysia facilitate various aspects of behavior. We now show that the chemosensory rhinophores sense these pheromones. Ablating the rhinophores causes a significant decrease in the time spent mating. In addition, the lesion blocks the increases of feeding in response to pheromones released by egg cordons and by mating conspecifics. Respiratory pumping is significantly increased in response to egg cordons, mating conspecifics and egg laying hormone (ELH). The increase in response to egg cordons is blocked by ablating the rhinophores, but not by lesioning the osphradium, a second chemosensory organ.

Coordination of Reproductive Activity in Aplysia: Peptide Neurohormones, Neurotransmitters, and Pheromones Encoded by the Egg-Laying Hormone Family of Genes.

Pheromones play a significant role in coordinating reproductive activity in the marine opisthobranch mollusk Aplysia. Although solitary during most of the year, these simultaneous hermaphrodites gather into breeding aggregations during the reproductive season. The aggregations contain both mating and egg-laying animals, and are associated with masses of recently deposited egg cordons. Behavioral studies suggest that cordon-derived pheromonal factors are primarily responsible for establishing and maintaining the aggregations. Egg-laying animals are more attractive than sexually mature, but nonlaying, conspecifics and have a shorter mean latency to mating; egg cordons and egg-cordon eluates, when placed in the surrounding seawater, enhance the attractiveness of nonlaying animals and reduce their mean latency to mating. Similar effects are observed when extracts of the atrial gland are placed in the seawater, suggesting that secretory products of this oviductal exocrine organ may function as sexual pheromones. Biochemical analyses indicate that there may be multiple attractants in atrial gland extracts, and that at least one of these (A-NTP) is a peptide encoded by the A gene. The A gene belongs to a small family of structurally related genes that are expressed in a tissue-specific manner. Another member of the family, the egg-laying hormone (ELH) gene, is expressed in the neuroendocrine bag cells. Peptide products of the ELH gene act as neurohormones and nonsynaptic neurotransmitters, initiating egg laying and coordinating its associated behaviors. Peptide products of a family of genes may thus act internally and externally to coordinate both male and female reproductive activities.

Common regulation of feeding and mating in Aplysia fasciata: Pheromones released by mating and by egg cordons increase feeding behavior

We examined whether pheromones released by reproductive behaviors (mating and egg-laying) affect feeding behavior. A preliminary experiment demonstrated that the quantity of food eaten can be used to measure the effects of pheromones on feeding. Using this measure, we then showed that Aplysia that were prevented from mating, but that were in the same aquarium as mating conspecifics, eat more food than do Aplysia in a medium lacking mating animals. Mating and feeding were not temporally correlated, indicating that pheromones released by mating probably do not initiate feeding, but rather modulate feeding after it has begun. Aplysia that were in the same aquarium as freshly deposited egg cordons also ate more than did animals in a medium lacking eggs.

Relative Contributions of the Egg Layer and Egg Cordon to Pheromonal Attraction and the Induction of Mating and Egg-laying Behavior in Aplysia.

Many species of the opisthobranch mollusk Aplysia form breeding aggregations during the reproductive season. The aggregations contain both mating and egg-laying animals, and are associated with egg cordons. Although pheromones play a significant role in developing and maintaining the aggregations, little is known about the active factors. Behavioral studies have shown that egg-laying animals are more attractive than nonlaying animals, have shorter latencies to mating, and induce conspecifics to lay eggs. As a first step toward isolating and chemically characterizing the active factors, we examined the relative importance of the egg layer and egg cordon as sources of pheromonal activity in Aplysia brasiliana. T-maze experiments showed that both animal-derived and cordon-derived factors are attractive, and that the animal-derived factors are specifically associated with egg layers. Extracts of the atrial gland--an exocrine organ secreting into the oviduct--increased the attractiveness of nonlaying animals when placed in the surrounding seawater, suggesting that the "cordon-derived" aggregation pheromones may be products of the atrial gland. Mating studies showed that both animal-derived and cordon-derived factors induce mating, and that the animal-derived factors are associated with both egg layers and nonlayers. In contrast, neither animal-derived nor cordon-derived factors induced egg laying. Comparable results were obtained with either one or two animals in the chamber, suggesting that the accessibility of a potential mate did not influence the results. The lack of effect may result from the low-probability nature of egg-laying activity.

Induction of copulatory behavior in Aplysia: Atrial gland factors mimic the excitatory effects of freshly deposited egg cordons

Egg laying in the marine mollusc Aplysia is induced and coordinated by peptide products of the egg-laying hormone (ELH) gene expressed in the neuroendocrine bag cells of the central nervous system. At least three structurally related genes, belonging to the ELH family but distinct from the ELH gene, are expressed in the atrial gland, an exocrine organ of unknown function that secretes into the oviduct of Aplysia. The experiments described in this report were designed to test the hypothesis that the atrial gland gene products serve a pheromonal function for the animal, coordinating reproductive behavior among individuals. Our studies showed that there was a significantly shorter latency to copulation when an Aplysia was paired with an animal that was actively laying eggs than when it was paired with a sexually mature but nonlaying animal. Moreover, the addition of extracts or homogenates of the atrial gland to the seawater surrounding two nonlaying animals reduced the latency to mating compared to animals exposed only to seawater or to homogenates of other regions of the reproductive tract, including oviduct. These results suggest that atrial gland products, secreted onto the egg cordon as it passes through the oviduct, may play a pheromonal role and induce mating behavior between individuals. Experiments are in progress to determine whether the active atrial gland factor(s) are products of the ELH-family genes expressed in the gland.

The anatomy and functional morphology of the large hermaphroditic duct of three species of Aplysia, with special reference to the atrial gland.

The anatomy and functional morphology of the large hermaphroditic duct of three species of gastropod mollusc (Aplysia californica, A. dactylomela, and A. brasiliana) were examined. Each duct is composed of two parallel compartments, the red hemiduct (RHD) and the white hemiduct (WHD), which are distinguishable from the outside of the duct. Four secretory regions, all exocrine in morphology, are recognizable: the RHD secretory epithelium, the atrial gland (or atrial gland-like epithelium), the WHD secretory epithelium, and the accessory gland of the copulatory duct (AGCD). Of these regions, only the atrial gland (or atrial gland-like epithelium) contains egglaying activity and only the atrial gland (or atrial gland-like epithelium) is immunocytochemically labeled by serum antibodies generated against low molecular weight A. californica atrial gland peptides. The RHD is the functional oviduct: the egg cordon passes through a channel lined by the RHD secretory epithelium and bordered by the atrial gland (or atrial gland-like epithelium); the eggs are separated from both the WHD secretory epithelium and the AGCD by internal folds of the duct. The WHD is the functional copulatory duct: the penis, exogenous sperm, and endogenous sperm pass directly by the AGCD and in close proximity to the WHD secretory epithelium; they are separated from both the RHD secretory epithelium and the atrial gland (or atrial gland-like epithelium) by internal folds. The atrial gland (or atrial gland-like epithelium) is thus not likely to have a prostatic function or to be directly stimulated by the penis during copulation; it may play a role in oviductal function.

Role of bag cells in egg deposition ofAplysia brasiliana

Egg deposition behaviors are analyzed from time-lapse recordings during which “spontaneous” discharges of the neuroendocrine bag cells are recorded with chronically implanted cuff electrodes. In the laboratory,Aplysia brasiliana normally deposit long egg cordons on the substrate in a characteristic “figure 8” pattern similar to the configuration of egg masses observed in the natural environment. The overt behaviors associated with egg deposition are rhythmic head movements consisting of three components that overlap with characteristic relative latencies: up-and-down “undulations”, side-to-side “weaves” and in-and-out “tamps”. The characteristics of the three behaviors and their time courses relative to the appearance of eggs on the substrate suggest that undulations prepare the substrate, weaves distribute the egg cordon and tamps attach the cordon to the substrate. The same rhythmic head movements are also elicited by injections of homogenized abdominal ganglia (HAG) containing bag cell clusters, with comparable “relative” latencies and maximum frequencies but for shorter total durations. The overt behaviors begin earlier for normal than for triggered egg laying, often before the “spontaneous” release of bag cell hormones. This suggests that the head oscillations in intact animals are not normally initiated by bag cell activity. The mean latency to the appearance of the egg cordon on the substrate is the same (about 34 min) following either HAG injections or spontaneous bag cell discharges, confirming previous suggestions that the bag cell discharge triggers ovulation. Furthermore, the head movements appear to terminate at the same time following release or injection of hormone. The accompanying paper demonstrates that the full expression of the behavioral effects of bag cell injections depend upon normal movement of eggs in the reproductive tract.