Direct innervation of the mantle edge gland by neurosecretory axons in Helisoma duryi (Mollusca: Pulmonata)

Summary The mantle edge gland of Helisoma duryi is innervated by neurosecretory axons from the pallial nerves. Synaptoid contacts occur between axons and gland cells, and there is ultrastructural evidence for the release of neurosecretory material. The mantle edge gland contributes to the deposition of periostracum during shell formation, and direct neurosecretory innervation may control shell growth and regeneration.Supported by a National Research Council of Canada Grant (A-4673) and Negotiated Grant D-61     

Cell junctions in the endocrine dorsal body cells of Helisoma duryi (Mollusca: Pulmonata): an in vitro study

The endocrine dorsal bodies of gastropod molluscs regulate reproduction and are closely associated with the central nervous system. Previous studies on Helisoma duryi have shown that the dorsal body cells of reproducing snails contain more gap junctions than those of non-reproducing virgin snails. More dorsal body cells were isolated from virgin snails than reproducing snails in Ca(2+) and Mg(2+) free experimental saline. The isolated spherical cells attached to the culture dish, spread and formed filopodium-like processes within a few hours in culture medium containing Ca(2+) and Mg(2+). Many isolated cells reaggregated after 4-6 h in culture forming septate junction-like and gap junction-like cell contacts, as revealed by thin section and freeze-fracture studies. Following 10 min incubation in carboxyfluorescein diacetate the isolated cells fluoresced and, after aggregation, these cells transferred fluorescent dye to unlabelled cells. Cell aggregation was inhibited by cytochalasin D. Staining by NBD-phallacidin revealed the presence of actin in the filopodium-like processes of spread cells and in the perinuclear cytoplasm. It is likely that the septate junction-like contacts provide sites of cell attachment between aggregating cells; gap junctions are involved in intercellular communication, and actin is required in this process.     

Morphological and immunohistochemical comparison of three rat prostate lobes (lateral, dorsal and ventral) in experimental hyperprolactinemia

The prolactin plays an important role in the regulation of growth and differentiation of prostate gland besides androgens. The goal of this study was to reveal the influence of elevated prolactin concentration on epithelial cells of prostate. We compared the morphology of epithelial cells of prostate dorsal, lateral and ventral lobes and expression of androgen receptors in these cells in rats with hyperprolactinemia and in control rats. We used sexually mature male Wistar rats. The experimental rats received metoclopramide; the control group received saline in the same way. The prostate dorsal, lateral and ventral lobes were collected routinely for light and electron microscopy. The intensity of immunohistochemical reaction of androgen receptor in epithelial cells of dorsal, lateral and ventral lobes was evaluated by measure of optical density with computer image analysis. The light and electron (transmission and scanning) microscopes were used for morphological observations. Results: In experimental rats twofold increase in prolactin and twofold decrease in testosterone found. In experimental group the expression of androgen receptor was lower in columnar epithelial cells of dorsal and ventral lobes but higher in lateral one. We observed morphological abnormalities in columnar epithelial cells of lateral and dorsal lobes. The columnar epithelial cells of ventral lobes didn't show any morphological changes in hyperprolactinemia.     

Cell contacts between follicle cells and the oocyte of Helisoma (Mollusca,Pulmonata)

The cell contacts between follicle cells, and follicle cells and oocytes of egg-laying populations of Helisoma duryi and non-egg-laying populations of H. trivcolvis have been studied. Scanning electron microscopy reveals that four to six follicle cells envelop a single developing oocyte. Thin sections and lanthanum impregnations demonstrate apical zonulae adherentes followed by winding pleated-type septate junctions between follicle cells. Gap junctions and septate junctions have been found between follicle cells and vitellogenic oocytes. Freeze-fracture replicas show relatively wide sinuous rows of septate junctional particles, and nemerous large gap junctional particle aggregates on the P-face between vitellogenic oocytes and follicle cells. Septate and gap junctions between immature or nonvitellogenic oocytes and follicle cells are fewer compared to those in vitellogenic oocytes. Similarly, the junctional complexes are less developed in non-egg-laying H. trivolvis compared to those in egg-laying H. duryi. It is possible that intimate interaction between follicle cells and a developing oocyte is necessary for the maturation of the oocyte. The junctional complexes could be involved in the interaction of the follicle cells and the oocyte, and they must disassemble at the onset of ovulation. Rhombic particle arrays and nonjunctional ridges of particles have been found in the basal part of the oolemma.     

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.     

Effects of Castration on Growth and Reproduction of Helisoma duryi (Mollusca: Pulmonata)

Summary

Castration of Helisoma duryi disturbs the balance between growth and reproduction in favour of growth, as is evidenced by the stimulation of body and shell growth and reduction in egg mass production when compared with sham operated controls. Female accessory sex organ wet weight is increased in castrated and virgin snails. Male accessory sex organ wet weight is unaffected and female accessory sex organ wet weight is reduced by dorsal body ablation, suggesting that growth of differentiated female accessory sex organs is stimulated by the endocrine dorsal bodies. Albumen gland synthetic activity is inhibited in the absence of a functioning gonad. This inhibition may be due partly to the accumulation of secretory products within the glands and partly to the inactivation of the endocrine dorsal bodies and neurosecretory caudodorsal cells in the cerebral ganglia. The circulating levels of the vitellogenic protein ferritin are reduced by castration, and increased by injection and implantation of active gonadal tissue into castrates. This may be due to an indirect effect of gonadal factor (s) on the extragonadal synthesis of ferritin.     

Neuronal pathways from the dorsal acelli of the house cricket,Acheta domesticus

Each ocellar nerve in the house cricket Acheta domesticus contains giant nerve fibers of 10-15 μ diameter, characterized in Golgi Cox preparations by a single row of short collaterals which runs along nearly the entire length of a fiber. Numerous long collaterals are given off by thin fibers in the ocellar nerve; medium-size fibers give off relatively few collaterals. The lateral ocellar tracts extend posteriorly through the dorsal protocerebrum, crossing the protocerebral bridge dorsally. The smaller median ocellar tract runs more ventrally through the pars intercerebralis; posterior to the bridge its fibers turn out toward the lateral nerves. Golgi and cobalt preparations reveal branching of giant and mediu_-size ocellar fibers posterior to the bridge at two levels, forming bilateral regions of ocellar neuropile. No ocellar processes appear to be given off to the corpora pedunculata, centra! body, nervi corporis cardiaci, antenna! lobes, or circumesophageal connectives; it is uncertain whether ocellar collaterals extend into the protocerebral bridge or optic lobes. Cell bodies of giant and medium-sized fibers are located in the pars intercerebralis.     

The cardiac innervation of Eledone cirrhosa (Lamarck) (Mollusca: Cephalopoda)

The innervation to the cardiac organs and vessels of the octopods Eledone cirrhosa, E. moschata and Octopus vulgaris is described from vitally stained fresh material and wax-embedded sections. This innervation arises from the paired visceral nerves and includes two main peripheral ganglia (fusiform and cardiac) on each side. Several new details of the innervation are reported. Nerves supplying the lateral venae cavae arise from the ventricular nerves at the level of the ventricle. Nerve fibres run to the efferent branchial vessels from the cardiac ganglia. A small ganglion, lying on the auriculo-ventricular nerve, is described for some specimens of both species of Eledone, and is named the auricular ganglion. Commissural strands linking the right and left ventricular nerves of either side are found in Eledone, comparable to those previously described from Octopus. The detailed branching pattern of the innervation shows considerable individual variation and consistent interspecific differences. In E. cirrhosa the fine fibres innervating the inner and outer muscle layers of the auricle show distinct differences in their configuration. Innervation at the surface of the ventricular lumen and around the coronary arterial vessels shows evidence of specialization. The muscle of the branchial heart, particularly the valve leaflets at the junction of the heart and the lateral vena cava, is abundantly innervated. The observations are discussed in relation to other cephalopods and to their probable physiological significance. It is suggested that they provide evidence for a greater degree of neural influence in the control of the cardiac organs than is usually supposed and that they support the idea that the lateral venae cavae have a significant role in the generation of circulatory pressures.     

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.     

Cloning and characterization of a candidate nutritive glycoprotein from the albumen gland of the freshwater snail, Helisoma duryi (Mollusca: Pulmonata)

Abstract. The albumen gland is a female accessory sex gland that synthesizes and secretes perivitelline fluid around pulmonate eggs. The perivitelline fluid is composed of mainly galactogen and proteins, and is thought to provide nourishment to the embryos during development. We have previously identified the major secretory protein of the albumen gland of the freshwater snail Helisoma duryi as a native glycoprotein of ∼288 kDa, consisting of four 66-kDa subunits. In this study, the major albumen gland protein in H. duryi was purified, cloned, and the full-length cDNA sequence determined. Nucleotide sequence analysis revealed that the albumen gland protein (HdAGP) shared 83% identity with a partial cDNA sequence from a developmentally regulated albumen gland protein in Biomphalaria glabrata . The HdAGP mRNA was detected by RT-PCR in the albumen gland, ovotestis, mantle and digestive gland. SDS-PAGE analysis of the albumen gland protein in egg masses at different stages of development showed that the amount of HdAGP steadily decreased during embryogenesis, suggesting its possible catabolism by the developing embryos. Protein domain searches suggested that the HdAGP shared limited sequence identity, and adopted a similar three-dimensional conformation to the bactericidal, permeability increasing, protein family, raising the possibility of a potential bactericidal function for this important reproductive/developmental protein.     

Convergent evolution of the lateral line system in Apogonidae (Teleostei: Percomorpha) determined from innervation

The lateral line system and its innervation were examined in two species of the family Apogonidae (Cercamia eremia [Apogoninae] and Pseudamia gelatinosa [Pseudamiinae]). Both species were characterized by numerous superficial neuromasts (SNs; total 2,717 in C. eremia; 9,650 in P. gelatinosa), including rows on the dorsal and ventral halves of the trunk, associated with one (in C. eremia) and three (in P. gelatinosa) reduced trunk canals. The pattern of SN innervation clearly demonstrated that the overall pattern of SN distribution had evolved convergently in the two species. In C. eremia, SN rows over the entire trunk were innervated by elongated branches of the dorsal longitudinal collector nerve (DLCN) anteriorly and lateral ramus posteriorly. In P. gelatinosa, the innervation pattern of the DLCN was mirrored on the ventral half of the trunk (ventral longitudinal collector nerve: VLCN). Elongated branches of the DLCN and VLCN innervated SN rows on the dorsal and ventral halves of the trunk, respectively. The reduced trunk canal(s) apparently had no direct relationship with the increase of SNs, because these branches originated deep to the lateral line scales, none innervating canal neuromast (CN) homologues on the surface of the scales. In P. gelatinosa, a CN (or an SN row: CN homologue) occurred on every other one of their small lateral line scales, while congeners (P. hayashii and P. zonata) had an SN row (CN homologue) on every one of their large lateral line scales.     

Immunocytochemical localization of FMRFamide in the central nervous system and the kidney of Helisoma duryi (Mollusca): its possible antidiuretic role

The distribution of FMRFamide-immunoreactive neurons in the central nervous system of the freshwater pulmonate, Helisoma duryi is described. All parts of the central nervous system except the two pleural and the right parietal ganglia, contain immunoreactive neurons. By immunogold techniques, only one kind of neurosecretory FMRFamide-immunoreactive cell (previously identified as the type-3 cell) was localized in the visceral and left parietal ganglia. This cell type has been previously implicated in an antidiuretic role. FMRFamide-immunoreactive material is found in the whole mount of the kidney as well as in kidney sections. Electron microscopic examination shows that the axons innervating either the smooth muscles of the kidney or the kidney itself contain neurosecretory granules morphologically similar to type-3 cells of the visceral and left parietal ganglia. When incubated in saline containing nanogram quantities of FMRFamide, the wet weight of the kidney increased. It is suggested that FMRFamide-like substance may function as an antidiuretic factor and that the kidney is a target organ of this peptide for osmoregulation.     

Pharmacological analysis of locomotion and heart contraction during the development of Helisoma (Mollusca: Gastropoda). Short communication

We investigated involvement of different 5-HT receptors in regulation of ciliary rotation, gliding locomotion and heartbeat of Helisoma embryo at pre- and post-metamorphic stages. Pharmacological analysis suggested that activation of 5-HT1 receptor enhance ciliary rotation but do not affect gliding locomotion. Activation of 5-HT4 receptor depresses both types of locomotion. Before metamorphosis heart contraction is depressed by activation of 5-HT4 and enhanced by activation of 5-HT7 receptor. However, the heart became insensitive to all agonists by hatching. We hypothesized that alterations in affinity or expression of particular 5-HT receptors can underlie the well-coordinated character of serotonin-dependent larval behavior.     

Evolutionary relationships among squids of the family Gonatidae (Mollusca: Cephalopoda) inferred from three mitochondrial loci

The oceanic squid family Gonatidae (Mollusca: Cephalopoda) is widely distributed in subpolar and temperate waters, exhibiting behavioral and physiological specializations associated with reproduction. Females of several species undergo muscular degeneration upon maturation; origins of this complex morphogenic change are unknown, hindering our understanding of ecological and morpho-physiological adaptations within the family. To provide further information regarding the evolutionary relationships within Gonatidae, three mitochondrial loci (12S rRNA, 16S rRNA, and cytochrome c oxidase subunit I) were analyzed for 39 individuals representing fourteen gonatid and six outgroup cephalopod species. In addition to elucidating relationships among gonatids, molecular data provided more information than morphological data for problematic specimens. Although some data sets are incongruent or have low nodal support values, combined molecular analysis confirms the presence of gonatid groups previously established by morphological characteristics (i.e., possessing radular teeth in seven longitudinal rows and muscular mantle tissue). These characteristics are basal to taxa possessing radular teeth in five longitudinal rows and less muscular mantle tissue, indicating that the derived forms are those species exhibiting physiological adaptation such as tissue degeneration upon maturation and egg brooding.     

The structure of the bursa copulatrix in virgin and mated snails, Helisoma duryi (Mollusca): role of acid phosphatase in reproduction

Abstract. The fine structure of the bursa copulatrix of the virgin snails has been compared with that of mated snails. One of the noticeable changes after mating is an increase in the number of the Golgi and the secretory vesicles. Since some of the vesicles react positively for acid phosphatase it is suggested that this enzyme activity increases following mating. The bursa lumen of the virgin snail contains gel-like materials devoid of spermatozoa, however, following mating, the lumen is full of semen containing live spermatozoa and bacteria. The source of bacteria in the lumen is not known. Acid phosphatase activity is significantly higher in the luminal content of mated snails than in the virgin snails. The activity is higher in the lumen than in the epithelial cells, suggesting that the enzyme is secreted into the lumen where it is utilized for extracellular degradation of spermatozoa. Following mating, the spermatozoa are motile in the lumen of the bursa for ∼3–7 d, but become immobile and finally undergo extracellular digestion so that intact spermatozoa are not recognizable by day 10. The use of castrated snails in mating experiments suggest that individuals of Helisoma duryi reproduce by cross fertilization and that the bursa may act as the holding organ from where the spermatozoa are periodically transported to the carrefour over ∼7 d. At day 10 following mating, however, autosperms appear in the hermaphroditic duct awaiting the next mating.     

Tissue inhibitor of metalloproteinase-2 (TIMP-2) location in the ventral, lateral, dorsal and anterior lobes of rat prostate by immunohistochemistry

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play a major role in extracellular matrix component degradation in several normal and abnormal tissue situations; they are also found in human seminal plasma. MMPs have been found in rat prostate secretions and are nearly lobe specific in expression pattern. The aim of this study was to evaluate whether TIMP-2, like other semen components, is expressed differently from different rat prostatic lobes. Immunohistochemical staining was performed in both young and adult rat ventral (VP), lateral (LP), dorsal (DP), and anterior (AP) prostatic lobes and confirmed by western blotting. TIMP-2 expression was found in the epithelial cells in the following sequence: LP>AP>DP>VP, in both young and adult rats. In this study, 100% of adult LP presented histological signs of prostatitis, where TIMP-2 immunostaining was positive in normal epithelium even with intraluminal neutrophils, but was reduced or absent in the epithelium with intraepithelial leukocytes or with periductal stroma disorganization associated with mononuclear cell infiltration. However, TIMP-2 expression in LP was not induced by prostatitis, since younger rat LPs were also strongly TIMP-2 positive. The distal and intermediate VP regions were TIMP-2 negative, but the proximal regions were strongly stained. Western blotting results confirmed the high TIMP-2 expression in the LP lobe. Thus, TIMP-2 is expressed differently between the prostatic lobes and is another nearly lobe-specific protein, which plays a role in the regulation of MMP activity in seminal plasma and glandular homeostasis. TIMP-2 is also another regional ductal variation of VP. Further studies should address whether TIMP-2 expression is related to the highest incidence of rat LP prostatitis and adenocarcinoma.     

Larval and early post-larval shell of three deep-sea Scaphopoda (Mollusca) from the southwest Atlantic

The larval shells of Antalis circumcincta (Watson, 1879) (order Dentaliida), Pertusiconcha callithrix (Dall, 1889) (order Gadilida) and of an undetermined species of uncertain systematic position are described. The material studied comprises mainly samples from deep waters, collected by expeditions along the southeast coast of Brazil. The larval shell of the three taxa matches other types previously described in the literature. Antalis circumcincta and P. callithrix have four regions (protoconch A, protoconch B, teleoconch A, teleoconch B), but differ in dimensions and sculpture from each other, while the undetermined species has three regions (protoconch A, protoconch B, teleoconch B). A morphometric approach combined with a discriminant analysis also indicates that the three taxa are significantly distinct. This study confirms patterns of larval shells at the taxonomic rank of orders but other supraspecific patterns remain uncertain.