Structure, localization and potential role of a novel molluscan trypsin inhibitor in Lymnaea.

Eggs and egg masses of the freshwater gastropod mollusc Lymnaea provide a microenvironment for developing embryos. Secretions of the exocrine albumen gland of Lymnaea are packaged in the eggs of an egg mass before the eggs are laid externally. The perivitelline fluid that directly surrounds individual oocytes is the main source of nutrition for developing embryos. During early stages of development, the perivitelline fluid is initially internalized by pinocytosis and degraded by lysosomes; in later stages, the embryo ingests the fluid. We previously found that the albumen gland produces large amounts of Lymnaea epidermal growth factor. The albumen gland also appears to produce significant amounts of a novel Lymnaea trypsin inhibitor (LTI), a second peptide that was purified and characterized from Lymnaea albumen gland extracts. The primary structure was determined by microsequence analysis, mass spectrometry, and C-terminal sequence analysis, and showed that LTI is a 57-residue glycosylated peptide. Comparison of the LTI sequence with other known serine protease inhibitors indicates that LTI is a member of the bovine pancreatic trypsin inhibitor family. Reverse phase-high performance liquid chromatography, microsequence analysis, mass spectrometry, and immunocytochemistry demonstrated that abundant amounts of intact LTI are packaged in egg masses. The presence of a trypsin inhibitor in the perivitelline fluid compartment of the egg mass may minimize digestion of peptides and proteins in the perivitelline fluid that are important for the development of the embryo, for example, Lymnaea epidermal growth factor.     

Maternal impact on egg development in Lymnaea stagnalis: a growth factor is produced by the albumen gland in the reproductive tract

Summary

In the freshwater snail Lymnaea stagnalis, perivitellin fluid is the main source of nutrition for developing embryos; it contains predominantly galactogen and proteins. The fluid is produced by the albumen gland, a large exocrine organ in the female reproductive tract. To further define the protein products of this gland and provide more information about the maternal contribution to egg development, albumen glands were extracted and the extracts purified by reversed-phase HPLC. One major HPLC peak fraction exhibited neurotrophic activity when bioassayed on identified Lymnaea neurons in vitro; it has been partially sequenced and has the highest degree of sequence identity with epidermal growth factor (EGF). These results demonstrate that a growth factor with neurotrophic activity is produced by the albumen gland, and is packaged along with the eggs to serve a growth-promoting function in the embryo.     

The role of calcium on protein secretion of the albumen gland in Helisoma duryi (Gastropoda)

Abstract. The albumen gland of the freshwater pulmonate snail Helisoma duryi produces and secretes the perivitelline fluid, which coats fertilized eggs and provides nutrients to the developing embryos. It is known that perivitelline fluid secretion is stimulated by dopamine through the activation of a dopamine D₁‐like receptor, which in turn stimulates cAMP production leading to the secretion of perivitelline fluid. This paper examines the glandular release of perivitelline fluid and provides evidence for the role of Ca²⁺ in the regulated secretion of perivitelline fluid based on protein secretion experiments and inositol 1,4,5‐trisphosphate assays. Dopamine‐stimulated protein secretion by the albumen gland is reduced in Ca²⁺‐free medium or in the presence of plasma membrane Ca²⁺ channel blockers, although the Ca²⁺ channel subtype involved is unclear. In addition, dopamine‐stimulated protein secretion does not directly involve phospholipase C‐generated signaling pathways and Ca²⁺ release from intracellular stores. Sarcoplasmic/endoplasmic reticulum Ca²⁺‐ATPase inhibitors had little effect on protein secretion when applied alone; however, they potentiated dopamine‐stimulated protein secretion. Dantrolene, an inhibitor of ryanodine receptors, 8‐(N,N‐diethylamino)‐octyl‐3,4,5‐trimethoxybenzoate hydrochloride, a nonspecific inhibitor of intracellular Ca²⁺ channels, and 2‐aminoethyldiphenylborate, an inhibitor of inositol 1,4,5‐trisphosphate receptors, did not suppress protein secretion, suggesting Ca²⁺ release from internal stores does not directly regulate protein secretion. Thus, the influx of Ca²⁺ from the extracellular space appears to be the major pathway mediating protein secretion by the albumen gland. The results are discussed with respect to the role of Ca²⁺ in controlling exocytosis of proteins from the albumen gland secretory cells.     

Biochemical Composition of the Eggs of the Freshwater Snail Lymnaea stagnalis and Oviposition-induced Restoration of Albumen Gland Secretion

Summary

Galactogen and protein form the main constituents of the eggs of Lymnaea stagnalis. The amount of galactogen per egg is fairly constant, irrespective of the size of the egg mass or the age of the snail.

The restoration of the albumen gland, which produces the perivitelline fluid for the eggs, was studied in long-day (16 hr light-8 hr dark) snails after spontaneous oviposition. The wet wt of the gland and its galactogen and protein contents are markedly increased within 8 hr and reach a maximum at 32 hr after oviposition. These maxima correspond to the levels determined in snails that did not lay eggs for at least 1 to 2 days. The amounts of galactogen and of protein in the albumen gland are linearly related to the wet wt of this gland.

The restoration period of the albumen gland almost covers the mean egglaying interval. This implies synchronized cycles of albumen storage and egg formation.

The estimated amount of galactogen, released by the albumen gland during egg mass formation, is in accordance with that deposited in the eggs. In contrast, the wet wt of the eggs is 4.6 times higher than that of the released secretory material. Since after oviposition water uptake by the eggs in the egg mass is negligible, the perivitelline fluid, which is released by the albumen gland and surrounds the egg cell, must be diluted in the reproductive tract of the snail prior to oviposition.     

Membrane transduction pathway in the neuronal control of protein secretion by the albumen gland in Helisoma (Mollusca)

The albumen gland in Helisoma secretes a perivitelline fluid which surrounds each egg and is made up of several 66 kDa protein subunits and polysaccharide complexes. Forskolin, an adenylate cyclase activator, stimulated the secretion and release of the perivitelline fluid. An acidic extract of the central nervous system increased the intracellular concentration of cAMP in the albumen gland and this results in the release of the 66 kDa molecule and other proteins. Digestion of the brain extract with proteases abolished this activity, suggesting that the factor is a peptide. Cyclic AMP analogues and [BMX also stimulated the protein secretion in dose-dependent manner. Forskolin when added with the brain factor had an additive response. SQ22536, a non-competitive inhibitor of adenylate cyclase, inhibited brain extract dependent adenylate cyclase activity whereas aluminum fluoride, a G protein activator, was found to stimulate adenylate cyclase. Dopamine also stimulates protein secretion by the albumen gland and through the application of various agonists and antagonists of dopamine, it was established that the neurotransmitter acts via D1-like receptors by stimulating adenylate cyclase.     

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.     

Release of proteins and polysaccharides from the albumen gland of the freshwater snail Helisoma duryi : effect of cAMP and brain extracts

The albumen gland is a compound tubular exocrine gland found in the female reproductive tract of freshwater pulmonate snails such as Helisoma duryi. It secretes a perivitelline fluid, composed of protein and polysaccharide complexes, and coats each fertilized egg. A 288-kDa native glycoprotein, composed of several 66-kDa subunits, was identified in soluble extracts of albumen gland. Forskolin stimulates the release of secretory granules, containing both proteins and polysaccharides, from the cytoplasm of the glandular cells. An acid extract of the central nervous system or the adenosine-3′,5′-cyclic monophosphate (cAMP) analogue 8-bromo cAMP, stimulates protein secretion from the gland. Pretreatment of the albumen gland with cAMP antagonist (R_p isomer of cAMP) inhibits the stimulatory effect of a brain extract. Digestion of brain extract with proteolytic enzymes abolishes its activity, suggesting the factor from the brain is peptidergic. The neuroactive agents serotonin, Phe-Met-Arg-Phe-amide, Tyr-Gly-Gly-Phe-Met-Arg-Phe-amide, small cardioactive peptide B, and caudodorsal cell hormone were also tested for potential secretion-promoting ability. Brain extracts were partially purified with a Sep-Pak C₁₈ reverse-phase cartridge and indicate the peptide is relatively hydrophobic. These results suggest that a brain peptide promotes the secretion of perivitelline fluid, and this is mediated by the adenylate cyclase/cAMP signal transduction pathway. Accepted: 19 December 1997     

Dopaminergic neurons in the brain and dopaminergic innervation of the albumen gland in mated and virgin helisoma duryi (mollusca: pulmonata)

Background

Dopamine was shown to stimulate the perivitelline fluid secretion by the albumen gland. Even though the albumen gland has been shown to contain catecholaminergic fibers and its innervation has been studied, the type of catecholamines, distribution of fibers and the precise source of this neural innervation has not yet been deduced. This study was designed to address these issues and examine the correlation between dopamine concentration and the sexual status of snails.

Results

Dopaminergic neurons were found in all ganglia except the pleural and right parietal, and their axons in all ganglia and major nerves of the brain. In the albumen gland dopaminergic axons formed a nerve tract in the central region, and a uniform net in other areas. Neuronal cell bodies were present in the vicinity of the axons. Dopamine was a major catecholamine in the brain and the albumen gland. No significant difference in dopamine quantity was found when the brain and the albumen gland of randomly mating, virgin and first time mated snails were compared.

Conclusions

Our results represent the first detailed studies regarding the catecholamine innervation and quantitation of neurotransmitters in the albumen gland. In this study we localized catecholaminergic neurons and axons in the albumen gland and the brain, identified these neurons and axons as dopaminergic, reported monoamines present in the albumen gland and the brain, and compared the dopamine content in the brain and the albumen gland of randomly mating, virgin and first time mated snails.     

First proteome of the egg perivitelline fluid of a freshwater gastropod with aerial oviposition

Pomacea canaliculata is a freshwater snail that deposits eggs on solid substrates above the water surface. Previous studies have emphasized the nutritional and protective functions of the three most abundant perivitelline fluid (PVF) protein complexes (ovorubin, PV2, and PV3) during its embryonic development, but little is known about the structure and function of other less abundant proteins. Using 2-DE, SDS-PAGE, MALDI TOF/TOF, and LC-MS/MS, we identified 59 proteins from the PVF of P. canaliculata, among which 19 are novel. KEGG analysis showed that the functions of the majority of these proteins are "unknown" (n = 34), "environmental information processing" (10), 9 of which are related to innate immunity, and "metabolism" (7). Suppressive subtractive hybridization revealed 21 PVF genes to be specific to the albumen gland, indicating this organ is the origin of many of the PVF proteins. Further, the 3 ovorubin subunits were identified with 30.2-35.0% identity among them, indicating their common origin but ancient duplications. Characterization of the PVF proteome has opened the gate for further studies aiming to understand the evolution of the novel proteins and their contribution to the switch to aerial oviposition.     

Effect of infection with Trichobilharzia ocellata and Schistosoma mansoni on the ultrastructure of the albumen gland of their respective hosts, Lymnaea stagnalis and Biomphalaria glabrata

The albumen gland, a female accessory sex gland of pulmonate snails, produces the perivitelline fluid. The ultrastructure of the albumen glands of control and infected specimens of Lymnaea stagnalis and Biomphalaria glabrata was studied. The albumen gland of L. stagnalis contains two types of secretory cells--light (active) and dark (inactive)--and two types of supporting cells--centroacinar and myoepithelial. The secretory cells apparently represent two activity stages of one type of cell. The gland B. glabrata possesses only one secretory cell type, which alternates with one type of supporting cell. The albumen glands of L. stagnalis and B. glabrata infected at a juvenile stage were studied 4 and 14 weeks (L. stagnalis) and 4 and 9 weeks (B. glabrata) after exposure. After four weeks' infection, B. glabrata produced some egg masses, but in subsequent stages egg mass production completely coased. Infected L. stagnalis never produced eggs. B. glabrata was apparently infected at a "physiologically" more mature stage than L. stagnalis. The morphology of the albumen glands four weeks after exposure (the daughter sporocyst stage) is in agreement with this hypothesis. At this interval the secretory cells of L. stagnalis appeared to be much more severely affected (inactive Golgi bodies and rough endoplasmic reticulum, crinophagy of the secretory granules) than the cells of B. glabrata. In the later stages studied (shedding of the cercariae), the glands of both species appeared to be completely inactive (reduced height of the epithelium, inactive organelles, crinophagy, absence of secretory granules). At this stage of infection, daughter sporocysts containing cercaria embryos were seen in the connective tissue of the albumen gland of B. glabrata, but not of L. stagnalis. The results thus indicate that the development and synthetic activity of the albumen gland are seriously affected by infection. These processes are known to be under the endocrine control of the female gonadotrophic hormones. Since it has been established that these hormones are normally present in the haemolymph of infected snails, the findings can be explained by assuming that the parasite interferes in some way or other with the snail's endocrine system.     

Structure of the female genital glands of the oviduct in the opisthobranch mollusc, Runcina

Runcina is a small hermaphroditic opisthobranch which possesses a monaulic reproductive system. In previous studies the male copulatory apparatus, the structure of the spermatophore and also the process of oogenesis have been described. The present paper gives an account of the ultrastructure of the female genital glands of the oviduct. In Runcina the oviduct comprises three primary regions, the albumen gland, the egg capsule gland and the mucous gland. Eggs enter the fertilization chamber and as they pass the opening of the albumen gland they become surrounded by albumen or perivitelline fluid. The eggs appear to become encapsulated as they traverse the egg-capsule gland and are eventually stuck together by mucus to form an egg mass. The epithelial lining of the three glands consists of alternating ciliated and secretory cells. The characteristics in secretory products of the glandular cells are described, and are discussed with reference to the way they contribute to egg vestment.     

Monoamines and their metabolites in the freshwater snail Biomphalaria glabrata

Metabolism of the major monoamines and their functions were studied in the freshwater snail Biomphalaria glabrata. In both juvenile and adult snails, the plasma (cell-free hemolymph) appears to act as a reservoir for most of these monoamines and their metabolites including among others, L-dopa and dopamine as major constituents. Significant quantities of L-tryptophan, precursor of indoleamines, also was found in the plasma. L-dopa, serotonin, homovanillic acid and dopamine were prominently represented in the central nervous system of the snail, while serotonin and its metabolites, 5-hydroxyindole acetic acid and 5-hydroxytryptophol were found in the ovotestis. Catecholamines such as L-dopa, dopamine and homovanillic acid were identified in the albumen gland. Functional aspects of both dopamine and serotonin were studied using in vitro cultures of albumen glands, the site of perivitelline fluid and galactogen synthesis in B. glabrata. Dopamine was found to stimulate the release of secretory proteins when exogenously added to gland cultures and this process was inhibited by chlorpromazine, a dopamine receptor antagonist. Similarly, exogenous serotonin stimulated in vitro protein secretion by albumen glands. Thus, these results suggest that monoamines may play important roles in regulating reproductive activity of this snail and provides an excellent model for studying neurotransmitter function and metabolism in molluscs.     

Changes in Osmotic Pressure and Swelling in Horseshoe Crab Embryos During Development

Water influx accompanying the swelling of embryos during normal development of horseshoe crabs, Limulus polyphemus and Tachypleus tridentatus , following a rupture of the chorion, was analyzed. The increase in volume of perivitelline fluid during deveopment was about 90 percent of the increase in total embryo weight. Considerable water discharge was observed on drying the embryos in air and a reversible water influx occurred with a second immersion in sea water, even though the embryos died as a result of this treatment. Since the osmotic pressure of the perivitelline fluids decreased markedly during development until the end of swelling, a close correlation between swelling and osmotic pressure was recognized. These results indicate that certain osmoactive substances may be produced in the perivitelline fluid at the initial stage of swelling.     

Egg Turning During Incubation has no Effect Upon the Growth of Embryos of Alligator mississippiensis

Alligator eggs are not turned during incubation, instead the embryo adheres to the top inside of the shell. Turning is alleged to shear off the embryo and kill it. Avian egg turning allegedly facilitates embryonic development by stimulating growth of the area vasculosa and minimizing the effects of unstirred yolk and albumen layers. From day 10 to day 45 of incubation, alligator eggs were experimentally turned, gently, through ± 60° in an hourly cycle. This turning regime killed only 6 out of 25 embryos. Compared with unturned controls, no significant effects were observed on the growth, production of extraembryonic fluids or utilization of albumen and yolk for those embryos that survived turning. The protein concentration of amniotic fluid at various stages of alligator development was examined in eggs incubated at 30 and 33°C. The fluid contained very little protein (max <8 mg) at any time: the protein concentration did not change consistently as development progressed. Differences in response to egg turning in birds and reptiles may be associated with the length of the incubation period, the protein content of the albumen and the mechanism of albumen utilization.     

Selective sequestration of an oviductal fluid glycoprotein in the perivitelline space of mouse oocytes and embryos

Previously, we identified a 215 kd glycoprotein, GP215, which is associated with postovulatory oocytes and embryos, but not with preovulatory oocytes (Kapur and Johnson, '85). In this paper a polyclonal antibody that specifically recognizes GP215 has been used to study the distribution of the molecule in association with ova and preimplantation embryos and in the female reproductive tract. GP215 is present in epithelial cells lining the cranial portions of the oviduct and in oviductal fluid, ovarian bursal fluid, and medium conditioned by oviductal tissue in vitro. Immunofluorescence assays of the ovum and early embryo show that GP215 is sequestered in the perivitelline space. Since preovulatory oocytes exposed to bursal fluid in vitro acquire GP215, we hypothesize that GP215 is synthesized and secreted by the oviductal epithelium and secondarily associates with the ovulated oocyte. Sequestration of GP215 within the perivitelline space is relatively specific since mouse serum albumin, a major constituent of oviductal fluid, and other high molecular weight proteins are not similarly retained. These observations indicate that the composition of the perivitelline space may be significantly different from the greater environment external to the zona pellucida such that fertilization and early development of mammalian ova potentially take place in a distinct perivitelline microenvironment.     

Analysis of albumen gland proteins suggests survival strategies of developing embryos of Pomacea canaliculata

The albumen gland of Pomacea canaliculata secretes the perivitelline fluid (PVF) surrounding the developing embryo which gives its eggs a highly effective defence against environmental factors and predators. Although previous studies have determined the functions of some PVF proteins extracted from P. canaliculata eggs, knowledge of the protein composition of PVF is still limited. In this study we use LC-MS/MS to identify 87 proteins from the albumen gland of P. canaliculata, 53 of which (60.9%) were annotated using the KEGG database. These classified into five functional groups: metabolism (54.7%); genetic information processing (9.4%); environmental information processing (3.8%); cellular processes (11.3%); and organismal systems (20.8%). The analyses found 12 proteins related to innate immunity and three proteins linked to defence against predation, providing important information for studies on embryo survival strategies and the invasive capabilities of P. canaliculata.     

Partial characterization of the secretory material from the dorsal bodies in the snail Helisoma duryi (Mollusca: Pulmonata), and its effects on reproduction

Abstract. Both dorsal body tissue extracts and dorsal body-conditioned medium stimulated in vitro polysaccharide synthesis in albumen gland explants in Helisoma duryi . This activity is heat- and protease-resistant. Dorsal body tissue extracts and dorsal body-conditioned medium were passed through solid-phase extraction cartridges, then eluted with increasing concentrations of methanol (20%, 70%, and 100%) and the various eluates tested for their biological activity. An active factor was found in the 100% methanol wash from both dorsal body tissue extracts and the conditioned medium. In addition, another bioactive factor in the conditioned medium eluted with 70% methanol. The endocrine dorsal bodies in the freshwater snail H. duryi were maintained in vitro , and following incubation, the culture medium was collected and tested for the presence of ecdysteroids. Radioimmunoassay of the culture medium demonstrated the presence of ecdysteroid-like immunoreactivity, suggesting the dorsal bodies are capable of secreting ecdysteroids in vitro . Identification of released ecdysteroids by HPLC/RIA revealed a number of immunoreactive fractions, which were tested for bioactivity. To test for possible physiological functions of ecdysteroids in Helisoma duryi , 20-hydroxyecdysone (a potent ecdysteroid in arthropods) was injected into non-egg laying (virgin) snails. Injections of ecdysteroid induced low egg-laying activity and the maturation of oocytes in the ovotestis. Incubation of albumen glands with ecdysteroid stimulated polysaccharide synthesis. The results are discussed in relation to the possible function(s) of ecdysteroids in pulmonate snails.