Comparative study on the metabolism of the androgen precursor androstenedione in two gastropod species: in vitro alterations by TBT and TPT

A comparative study was performed to assess the metabolism of the androgen precursor androstenedione (AD) in two gastropod species from the Muricidae family: Bolinus brandaris and Hexaplex trunculus. AD was mainly converted to 5alpha-dihydrotestosterone by microsomal fractions isolated from Bolinus brandaris, whereas it was primarily metabolized to testosterone by Hexaplex trunculus. Sex differences in the metabolism of AD were only detected in Bolinus brandaris and attributed to higher 5alpha-reductase activity in males. Thereafter, the effect of the organotin compounds, tributyltin (TBT) and triphenyltin (TPT), on the metabolism of AD was investigated. A significant interference was only detected in females, and differences between the modes of action of the two compounds were observed: TPT was a strong inhibitor of 5alpha-reductase activity in B. brandaris at a concentration as low as 100 nM whereas only TBT (10 microM) altered the metabolism of AD in H. trunculus by increasing the activity 17beta-hydroxysteroid dehydrogenase (17beta-HSD). Thus, this work shows that the metabolism of the androgen precursor AD strongly differs among gastropod species, both in terms of activity and metabolic profile, and further demonstrates the ability of TBT and TPT to interfere with key enzymatic pathways involved in androgen synthesis.     

Cytogenetics of Bolinus brandaris and phylogenetic inferences within the Muricidae (Mollusca: Gastropoda)

The purple dye murex, Bolinus brandaris (Linnaeus, 1758), is a muricid gastropod common throughout the Mediterranean and along the Moroccan and Portuguese Atlantic coasts. In the present study, we confirmed the diploid chromosome number of 2 n  = 70 for this species, and established for the first time the karyotype, which comprised 12 metacentric, 15 submetacentric and eight subtelocentric chromosome pairs. To facilitate cytotaxonomic comparisons, we carried out a comparative karyological analysis through multidimensional scaling between B. brandaris and three other 2 n  = 70 muricid species ( Hexaplex trunculus , Ocenebra erinaceus , and Stramonita haemastoma ) for which chromosomal measurements have been previously published. The interpretation of the ideograms and the statistical analysis highlighted the closest similarity of B. brandaris and H. trunculus compared to S. haemastoma and O. erinaceus . Indeed, B. brandaris and H. trunculus showed the smallest dissimilarities both for relative length and arm ratio, with O. erinaceus presenting intermediate values, whereas the highest dissimilarities were found between H. trunculus and S. haemastoma for both data. The karyotypes of B. brandaris and H. trunculus (subfamily Muricinae) presented the highest proportions of metacentric chromosomes compared to the other two muricids analysed, suggesting that those karyotypes could be considered primitive within the 2 n  = 70 Muricidae studied so far.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 185–193.     

Atypical spermatogenesis in Murex brandaris

Atypical spermatogenesis in Murex brandaris occurs in specific areas of the testis. In the early stages, a large nucleus is apparent, together with highly electrodense drops of chromatin spread through the caryoplasm. Many mitochondria are observed at one point of the cytoplasm, and numerous centrioles appear. In the following stages, vacuolization and degeneration of the chromatin are apparent, producing half moon-shaped chromatinic bodies called "caryomerites." Some degeneration of the chondrioma occurs too. Later, the chromatin degenerates and the centrioles produce cilia, while the Golgi body, highly secretory at this stage, produces electrodense PAS- as well as Thiery-positive granules. Moreover, a vermiform-shaped anuclear ciliated cell with numerous electrodense cytoplasmic granules is observed in the M. brandaris atypical spermatozoon.     

Ultrastructural study of the mucosa of the male gonoduct of Murex brandaris (Hexaplex brandaris) (Gastropoda,Prosobranchia)

Summary

The male gonoduct of Murex brandaris (Hexaplex brandarte) is covered by a columnar ciliated epithelium in which some goblet cells are located at the first level of the gonoduct. The structure of these cells suggests a transport and secretory epithelium. Some differences in ciliary organization and in cellular junctions are shown. The presence of some abnormal cilia is also detected.     

N-acetylmuramic acid in mollusca gastropoda: a histochemical and immunocytochemical study

Summary The presence of N-acetylmuramic acid in glycoconjugates in various mollusc tissues was investigated by histochemical and immunocytochemical techniques. The tissues studied included foot, mantle, digestive gland, ganglia and haemocytes ofHelix aspersa, Planorbarius corneus, Murex brandaris andTrunculariposis trunculus. Sialic acid residues were found to be absent. The possibility that N-acetylmuramic acid replaces sialic acid in acid glycoconjugates of gastropods with similar properties is discussed.     

Changes in nuclear structure during eupyrene spermatogenesis in Murex brandaris

Changes in nuclear structure during eupyrene spermatogenesis of Murex brandaris have been studied using light and electron microscopy. In the first phases, spermatogonia show round nuclei, with several electrodense masses of chromatin and a thin layer of heterochromatin associated with the nuclear membrane. Primary spermatocytes possess larger nucleii, with less condensed chromatin, and the synaptonemal complexes are apparent. During spermiogenesis, chromatin becomes lamellar, and the nucleus twists about its principal axis while it elongates. The nuclear twisting is accompanied by a progressive chromatin condensation, which causes a highly electrodense nucleus at the end of the process.     

Possible mechanisms for early and intermediate stages of sperm chromatin condensation patterning involving phase separation dynamics

During spermiogenesis in some internally fertilizing molluscs and insects, the post-meiotic spermatid nucleus develops via a sequence of complex patterns of the nuclear contents (chromatin and nucleoplasm) on the way to final chromatin condensation. We have examined the TEM data on these sequences for three species: Philaenus spumarius(a homopteran insect), Murex brandaris (a gastropod mollusc), and Eledone cirrhosa(a cephalopod mollusc). For each of these, spatially quantitative study reveals a constant spacing between pattern repeats through changes from granular to fibrillar to lamellar pattern, followed finally by a shrinkage of the spacing. Therefore we distinguish a "patterning" stage followed by a "condensation" stage. The former appears to demand a dynamic explanation, because there is no sign of structural connections to establish the part of the spacing that crosses the nucleoplasm. We consider types of dynamic mechanism, and show that for "nanostructural" dimensions (tens of nanometers as pattern spacing) reaction-diffusion dynamics are quite inappropriate, but that separation of two fluid phases by a mechanism similar to what is known as "spinodal decomposition" is a very attractive possibility.     

Mytilus inhibitory peptides (MIP) in the central and peripheral nervous system of the pulmonate gastropods, Lymnaea stagnalis and Helix pomatia: distribution and physiological actions

The distribution and neuroanatomy of Mytilus inhibitory peptides (MIP)-containing neurons in the central nervous system and their innervation pattern in the peripheral nervous system of the pulmonate snail species, Lymnaea stagnalis and Helix pomatia, have been investigated immunocytochemically, by applying an antibody raised to GSPMFVamide. A significant number of immunoreactive neurons occurs in the central nervous system of both species (Lymnaea: ca 600-700, Helix: ca 400-500), but their distribution is different. In Lymnaea, labeled neurons are found in all central ganglia where a number of large and giant neurons, previously identified physiologically, reveal MIP immunoreactivity. In Helix, most of the immunolabeled neurons are small (12-30 microm) and concentrated in the buccal and cerebral ganglia; the parietal ganglia are free of labeled cells. In both species, the ganglionic neuropils, peripheral nerves, connectives, and commissures are richly supplied with immunolabeled fibers. The MIP-immunoreactive innervation pattern in the heart, intestine, buccal mass and radula, and foot is similar in both species, with labeled axonal bundles and terminal-like arborizations (buccal mass, foot) or a network of varicose fibers (heart, intestine). Intrinsic neurons are not present in these tissues. The application of GSPYFVamide inhibits the spontaneous contractions of the esophageal longitudinal musculature in Helix, indicating the bioactivity of the peptide. An outside-out patch-clamp technique has demonstrated that GSPYFVamide opens the K+ channels in central nerve cells of Helix. Injection of GSPYFVamide into the body cavity inhibits the feeding of starved Helix. A wide modulatory role of MIP at central and peripheral levels is suggested in Lymnaea and Helix, including the participation in intercellular signalling processes and remote neurohormonal-like control effects.     

Diurnal rhythms in cyclic nucleotide metabolism in Helix nervous system.

Concentrations of cAMP (cyclic adenosine 3',5'-monophosphate) and cGMP (cyclic guanosine 3',5'-monophosphate), in ganglia from the garden snail Helix pomatia, vary considerably over the course of the day. There is a maximum in the concentration of both cyclic nucleotides between 08:00 and 12:00 (lights on 06:00 to 18:00), with the cAMP maximum occurring slightly later than that in cGMP. In addition there can be several smaller maxima in cAMP and cGMP levels; the timing of these can be markedly different from experiment to experiment, with cAMP and cGMP sometimes in and sometimes out of phase with each other. This pattern is observed in Helix which had been activated from the dormant state 4-6 days earlier, but is not present in dormant or in long-active animals. The cyclic nucleotide rhythm can be seen in ganglia maintained in organ culture, and persists for at least 24 hours after removal of the tissue from the animal. There appears to be little change in the level of basal or NaF-stimulated adenylate cyclase activity in Helix ganglia over the course of the day. On the other hand, both cAMP and cGMP phosphodiesterase activities exhibit rhythms which are consistent with the rhythms in cAMP and cGMP concentrations.     

The presence and specificity of crustacean cardioactive peptide (CCAP)-immunoreactivity in gastropod neurons

CCAP-like immunoreactivity was detected in central neurons with small and medium diameters in both Helix and Lymnaea CNS. The intensity of immunoreactivity showed seasonal changes with a maximum intensity during spring. The overwhelming majority of nerve cell bodies exhibiting CCAP immunoreactivity is located in the cerebral and parietal ganglia of both Helix and Lymnaea. The neurons of pleural and buccal ganglia were devoid of CCAP-immunoreactivity. Following preabsorbtion of CCAP antibody in 1:15000 dilution with 10(-3) M CCAP or CCAP-related peptide (Helix -CCAP), immunoreactivity could not be observed in neurons, demonstrating the specificity of the antibody to CCAP-related molecules in both Helix and Lymnaea.     

ADENYLATE CYCLASE IN HELIX AND APLYSIA GANGLIA: CHARACTERISTICS OF ITS STIMULATION BY A PEPTIDE-CONTAINING NERVOUS SYSTEM EXTRACT

Abstract— A crude particulate fraction, prepared from the central ganglia of Helix or Aplysia , contains levels of adenylate cyclase activity comparable to those in mammalian brain. This activity can be stimulated up to 50-fold by NaF, and 4- to 10-fold by guanyl nucleotides such as GTP and guanylylimidodiphosphate (Gpp(NH)p). A peptide-containing extract from Helix or Aplysia nervous system also stimulates the adenylate cyclase, by 50-400°. In contrast, a number of peptides known to occur in vertebrate and invertebrate nervous system are without effect. The adenylate cylase stimulation by the endogenous molluscan peptide-containing extract may be receptor-mediated, but the effect is not enhanced in the presence of guanyl nucleotides: in this respect it differs from many other hormone-sensitive adenylate cyclases. The endogenous extracts prepared from Helix and Aplysia each stimulate both Helix and Aplysia adenylate cyclases, suggesting that the putative cyclase-linked receptors may be similar in the two species. Furthermore, the active components in the extracts from Helis and Aplysia appear to be similar, since preliminary evidence suggests that they may interact with the same adenylate cyclase-linked receptor in particulate fraction from Helix ganglia.     

Distribution of alpha CDCP-immunoreactive neurons in the central nervous system of the snail Helix aspersa.

alpha CDCP is a neuropeptide produced by the caudodorsal cells of Lymnaea stagnalis and encoded by the genes of the egg-laying hormone (ELH). The use of a polyclonal antiserum raised against alpha CDCP resulted in the detection of about 800 immunoreactive neurons in the parietal ganglia and a small population (60 cells) in the cerebral ganglia of Helix aspersa. As the genes of ELH are well conserved among the gastropod species, these data designate the parietal ganglia as a putative source for the egg-laying hormone in Helix aspersa.     

Characterization of chromatin-condensing proteins during spermiogenesis in a neogastropod mollusc (Murex brandaris)

During the process of chromatin cndensation in the spermiogenesis of the neogastropod mollusc Murex brandaris, the nuclear protein complement undergoes a complex series of changes. These changes lead to the appearance of three small protamines in the ripe sperm nuclei. We have characterized this system electrophoretically and at the compositions with antibodies elicited against a specific spermatozoan protamine. Our results indicate that the complex pattern of chromatin condensation during spermiogenesis in this species (M. brandaris) may be modulated by a series of post-translational (and intranuclear) modifications of DNA-interacting proteins, such as precursors to the sperm protamines. The amino acid composition of each sperm protamine is remarkably simple (lys + arg + gly ≥96 mol%). This system of spermiogenic/spermatozoal proteins in the neogastropod M. brandaris clearly differs from that in patellogastropods and archaeogastropods, and it may be helpful in understanding evolutionary changes in the chromatin condensation pattern during the spermiogenesis of gastropod molluscs. © 1994 Wiley-Liss, Inc.     

High resolution spatial distribution of neuropeptides by MALDI imaging mass spectrometry in the terrestrial snail, Helix pomatia

Imaging mass spectrometry (IMS) is a powerful technique that combines the chemical and spatial analysis of surface materials. It allows spatial localization of peptides, proteins or lipids that are recorded in parallel without the need of a label. It is currently one of the most rapidly developing techniques in the proteomics toolbox. In the present study, accurate mass matrix-assisted laser desorption/ionization imaging mass spectrometry (MALD IMS) was used for direct molecular mapping of nervous tissue at micrometer spatial resolution. Cryosections of the whole brain of the terrestrial snail, Helix pomatia, were placed on indium-tin-oxide (ITO)-coated conductive glass slides and covered with a thin layer of α-cyano-4-hydroxycinnamic acid (CHCA) matrix by electro spray deposition. High-resolution molecular ion maps of well-known neuropeptides, such as FMRFamide were constructed. FMRFamide is known to exert powerful modulatory effect on synaptic transmission in molluscs. FMRFamide was predominantly localized in the cluster of neurons in the pro-, meso- and postcerebral regions of cerebral ganglia, pedal ganglia and right parietal ganglia of the central nervous system. Our present study, using MALDI IMS confirmed the distribution of FMRFamide containing cells in the Helix central nervous system previously detected by antibody dependent immunohistochemistry.     

Characterization by immunocytochemistry of ionic channels in Helix aspersa suboesophageal brain ganglia neurons

The aim of this work was to characterize several ionic channels in nervous cells of the suboesophageal visceral, left and right parietal, and left and right pleural brain ganglia complex of the snail Helix aspersa by immunocytochemistry. We have studied the immunostaining reaction for a wide panel of eleven polyclonal antibodies raised against mammal antigens as follows: voltage-gated-Na+ channel; voltage-gated-delayed-rectifier-K+ channel; SK2-small-conductance-Ca2+-dependent-K+ channel apamin sensitive; SK3 potassium channel; charybdotoxin-sensitive voltage-dependent potassium channel; BKCa-maxi-conductance-Ca2+-dependent-K+ channel; hyperpolarization-activated cyclic nucleotide-gated potassium channel 4; G-protein-activated inwardly rectifying potassium channel GIRK2 and voltage-gated-calcium of L, N and P/Q type channels. Our results show positive reaction in neurons, but neither in glia cells nor in processes in the Helix suboesophageal ganglia. Our results suggest the occurrence of molecules in Helix neurons sharing antigenic determinants with mammal ionic channels. The reaction density and distribution of immunoreactive staining within neurons is specific for each one of the antisera tested. The studies of co-localization of immunoreaction, on alternate serial sections of the anterior right parietal ganglion, have shown for several recognized mapped neurons that they can simultaneously be expressed among two and seven different ionic protein channels. These results are considered a key structural support for the interpretation of Helix aspersa neuron electrophysiological activity.     

Distribution of serotonin-containing neurons in the central nervous system of the snail Helix pomatia

Summary The distribution of serotonin (5HT)-containing neurons in the central nervous system of the snail Helix pomatia has been determined in whole-mount preparations by use of immunocytochemical and in vivo 5,6-dihydroxy-tryptamine labelling. 5HT-immunoreactive neuronal somata occur in all but the buccal and pleural ganglia. Immunoreactive fibres are present throughout the central nervous system. The 5HT-immunoreactive neuronal somata characteristically appear in groups, located mainly in the cerebral, pedal, visceral and right parietal ganglia. The majority of 5HT-immunoreactive neurons is located in the pedal ganglia. Additionally a dense network of 5HT-immunoreactive varicose fibres is found in the neural sheath of the central nervous system including all the nerves and ganglia. The number and distribution of 5HT-immunoreactive neurons correlates with that demonstrated by 5,6-dihydroxytryptamine labelling method.     

PHOSPHOLIPIDS IN THE NERVOUS SYSTEM OF MOLLUSCS

Abstract The phospholipid composition of nervous ganglia of the bivalve Unio crassa , the gastropod Helix pomatia and the cephalopods Octopus sp. and Ommastrephes sloanei pacificus have been investigated.
The ganglia of cephalopods contain considerably more phospholipids than do gastropod and bivalve ganglia. Especially rich in phospholipids are the optic ganglion of the squid Ommastrephes and the cerebral ganglion of Octopus , where their content is of the same order as in the brain of teleosts and amphibia.
In the ganglia of the lower molluscs, the bivalve and the gastropod, no sphingomyelin nor X-phospholipid could be detected.
No sphingomyelin nor X-phospholipid were found in the optic ganglion of Octopus , whereas in its cerebral ganglion sphingomyelin but no X-phospholipid was present.
In both the optic and the cerebral ganglia of the squid Ommastrephes both sphingomyelin and X-phospholipid were found.     

Diurnal rhythms in cyclic nucleotide metabolism in Helix nervous system

Concentrations of cAMP (cyclic adenosine 3′,5′-monophosphate) and cGMP (cyclic guanosine 3′,5′-monophosphate), in ganglia from the garden snail Helix pomatia, vary considerably over the course of the day. There is a maximum in the concentration of both cyclic nucleotides between 08:00 and 12:00 (lights on 06:00 to 18:00), with the cAMP maximum occurring slightly later than that in cGMP. In addition there can be several smaller maxima in cAMP and cGMP levels; the timing of these can be markedly different from experiment to experiment, with cAMP and cGMP sometimes in and sometimes out of phase with each other. This pattern is observed in Helix which had been activated from the dormant state 4–6 days earlier, but is not present in dormant or in long-active animals. The cyclic nucleotide rhythm can be seen in ganglia maintained in organ culture, and persists for at least 24 hours after removal of the tissue from the animal. There appears to be little change in the level of basal or Na Fstimulated adenylate cyclase activity in Helix ganglia over the course of the day. On the other hand, both cAMP and cGMP phosphodiesterase activities exhibit rhythms which are consistent with the rhythms in cAMP and cGMP concentrations.     

Localization of connexins in neurons and glia cells of the Helix aspersa suboesophageal brain ganglia by immunocytochemistry

The aim of the present study was to examine the distribution of cells expressing connexin 26 (Cx26) in the suboesophageal visceral, left and right parietal and left and right pleural ganglia of the snail Helix aspersa by immunocytochemistry. Altogether we have found approximately 452 immunoreactive neurons which represent the 4.7% of the total neurons counted. The stained large neurons (measured diameter 55-140 microm) occurred mostly on the peripheral surface of the ganglia while the small immunostained cells (5-25 microm diameter) were observed in groups near the neuropil. The number of large neurons giving positive Cx26-like immunostaining was small in comparison with that for medium (30-50 microm diameter) and small sized cells. The expression of Cx26 was also observed in the processes of glia cells localized among neurons somata and in the neuropil showing that the antiserum recognized epitopes in both protoplasmic and fibrous glia cells of Helix aspersa. The neuropils of all ganglia showed fibers densely immunostained. While we have observed a good specificity for Cx26-antiserum in neurons, a lack of reaction for Cx43 antiserum was observed in neurons and glia cells. The reaction for enolase antiserum in neurons was light and non-specific and a lack of reaction in glia cells and processes for GFAP antiserum was observed. Although the percentage of positive neurons for Cx26 antiserum was low is suggested that in normal physiological conditions or under stimulation the expression of connexin could be increased. The observed results can be considered of interest in the interpretation of Helix aspersa elemental two neuron networks synchronizing activity, observed under applied extremely low frequency magnetic fields.