Biosynthesis and axonal transport of multiple molluscan insulin-related peptides by the neuroendocrine light green cells of Lymnaea stagnalis.

The neuroendocrine light green cells (LGCs) control body growth and metabolism of the freshwater snail, Lymnaea stagnalis. These cells, located in the cerebral ganglia, use the peripheries of the median lip nerves (MLNs) as the neurohemal area. The LGCs express four members of a gene family encoding the precursors of distinct though related molluscan insulin-related peptides (MIPs). The molecular characterization and biosynthesis of MIPs have been investigated. MLN extracts were first size fractionated using gel permeation chromatography, and a major peak that coeluted with bovine insulin was further resolved using reverse phase high performance liquid chromatography. Amino acid composition analysis revealed that the four MIPs were present, as encoded by the MIP genes. Biosynthesis and axonal transport of the MIPs were studied in vitro with LGC systems in a series of pulse-chase incubations in combination with gel permeation chromatography analysis. During the pulse period of 20 min MIP precursors were formed in the LGC cell bodies. During the subsequent chase period these precursors were converted to MIPs and C peptides, which were transported to the LGC axon terminals in the MLNs for storage and release. The MIP precursors seem to be processed similarly to the insulin precursor in pancreatic islets.

Neuropeptide schistosomin inhibits hormonally-induced ovulation in the freshwater snail Lymnaea stagnalis.

This study examines the interaction between the caudodorsal cell hormone (CDCH) and schistosomin, a peptide secreted by the central nervous system of the snail (Lymnaea stagnalis) infected with the avian schistosome Trichobilharzia ocellata. Non-infected snails were injected with synthetic as well as native CDCH in the absence or presence of purified schistosomin. The response to 2 pmol of synthetic CDCH was blocked for 90% by coinjection with 3.5 pmol of schistosomin. The ovulation-inducing activity of extracts of cerebral commissures (the storage area of native CDCH) was also blocked by schistosomin. The degree of inhibition (65%), however, was less than that observed with synthetic CDCH. These results show that schistosomin inhibits ovulation and egg laying in Lymnaea. This explains the decrease or absence of egg laying in schistosome-infected freshwater snails.

The brain of Lymnaea contains a family of FMRFamide-like peptides.

Authentic FMRFamide and two FMRFamide-related heptapeptides were purified from the central nervous system of the fresh water snail Lymnaea stagnalis. The sequences of the heptapeptides were determined as: Ser-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (SDPFLRFamide) and Gly-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (GDPFLRFamide) by modified Edman degradation and enzymatic digestion. Relatively high quantities of the deamidated and therefore non-immunoreactive analogs of these two peptides (SDPFLRF and GDPFLRF) were also found. SDPFLRFamide and GDPFLRFamide were synthesized and were found to be chromatographically and biologically indistinguishable from the natural peptides, confirming the sequences. The log dose-response curves for the chronotropic action of either synthetic peptide on the heart of Lymnaea was very similar to that of FMRFamide. These data indicate that Lymnaea contains a family of FMRFamide-like peptides.

Purification and amino acid sequence of the ovulation neurohormone of Lymnaea stagnalis.

The neurosecretory caudodorsal cells of the freshwater pulmonate snail Lymnaea stagnalis produce an ovulation hormone [caudodorsal cell hormone (CDCH)] that is stored and released at the periphery of the intercerebral commissure. In the present study, CDCH has been purified and sequenced by micromethods. CDCH has been isolated, starting with a hydrochloric acid extract of commissures, by chromatofocusing, by high-performance, gel-permeation chromatography, and by reversed-phase, high-performance liquid chromatography. This procedure resulted in a 1690-fold purification and a 66% recovery. The data of the sequence analysis of CDCH are in agreement with the amino acid composition and reveal the following sequence of 36 amino acids: H-Leu-Ser-Ile-Thr-Asn-Asp-Leu-Arg-Ala-Ile-Ala-Asp-Ser-Tyr-Leu-Tyr-Asp-Gln-His -Trp-Leu-Arg-Glu-Arg-Gln-Glu-Glu-Asn-Leu-Arg-Arg-Arg-Phe-Leu-Glu-Leu-OH. Enzyme data indicate that the COOH end of the hormone is amidated. CDCH has a calculated isoelectric point of 9.0 and a calculated M(r) of 4529. CDCH shares a 44% homology with the sequence of the egg-laying hormone of the marine opisthobranch mollusc Aplysia californica.

Environmental and hormonal control of the seasonal egg laying period in field specimens of Lymnaea stagnalis.

A study has been made in field specimens of Lymnaea stagnalis of the relationship between environmental factors (temperature, photoperiod, and food) and spontaneous ovipository activity as well as oviposition evoked by injection of the ovulation hormone (CDCH) during a 1-year cycle. It appeared that spontaneous egg laying started in mid-May and ended in mid-September. It is concluded that it is the quantity of assimilated food that triggers the onset of the egg laying season. Its termination is very likely determined by a synergistic action of environmental factors. It is suggested that these factors control the activities of the CDCH-producing neuro-endocrine caudo-dorsal cells. In winter the snails are completely insensitive to injected CDCH, although many large oocytes are present in the gonad. During 2 months before and after the egg laying season, however, many injected snails respond to CDCH. It is argued that these phenomena are caused by changes in the activities of the endocrine dorsal bodies, which control vitellogenesis and the activities of the female accessory sex organs. The changes in the activities of the dorsal bodies are probably controlled by the synergistic actions of the previously mentioned environmental factors. Oviposition latency (interval between CDCH injection and start of oviposition) is much shorter during the egg laying season than in the nonreproductive period. This must be ascribed to the direct effects of temperature.

Ovulation hormone, nutritive state, and female reproductive activity in Lymnaea stagnalis.

A study of the relation between nutritive state and female reproductive activity as affected by the ovulation hormone (CDCH) has been made in the freshwater pulmonate snail Lymnaea stagnalis. CDCH is produced by the neuroendocrine caudodorsal cells (CDC) in the cerebral ganglia. Spontaneous oviposition ceased within 6 days of the beginning of a starvation period. This is most probably partially due to a reduction in the CDC activities because (1) quantitative electron microscopy showed a nearly 80% decrease in the number of release phenomena in the CDC-axon terminals in the neurohemal area in the intercerebral commissure, and (2) a bioassay showed a considerable reduction in the amount of CDCH in this area. During starvation the ovotestis and the female accessory sex organs became progressively less sensitive and, after 25 days, were completely insensitive to injected CDCH. This was indicated by a decrease in the responses to CDCH injection and, correspondingly, by a gradual increase in the CDCH thresholds for ovulation and egg formation. It is argued that the insensitivity may be caused by a reduction in the activities of the endocrine dorsal bodies. During refeeding, CDCH injections again become effective in inducing egg mass production, followed by resumption of spontaneous oviposition. This suggests a rapid restoration of DB and CDC activities following refeeding.

Synchronous maturation of the female reproductive system and of the ovulation hormone-producing system in Lymnaea stagnalis.

The intercerebral commissures (COMs) of young snails, which are in the male stage (10-15 mm shell height), contain only small quantities of ovulation hormone (CDCH): 0.6 OIU . COM-1 . g-1 snail (1 ovulation inducing unit = threshold dose for ovulation in adult recipients). During the subsequent period of female maturation the quantities of CDCH increase considerably up to a maximum of 47 OIU . COM-1 . g-1 snail at the onset of oviposition. In snails smaller than ca. 21 mm shell height oviposition cannot be induced by COM extract injections. It is concluded that maturation of the female reproductive system and of the ovulation hormone system occur simultaneously, and that both processes are stimulated by the hormone of the dorsal bodies (endocrine organs located upon the cerebral ganglia).

Specificity of ovulation hormones of some basommatophoran species studied by means of iso- and heterospecific injections.

Isospecific injections of intercerebral commissure (COM) extracts of L. stagnalis, L. palustris, and L. ovata (Lymnaeidae) and of extracts of COM/mediodorsal bodies complexes (COM/DB) of Bulinus truncatus (Bulinidae) and Biomphalaria glabrata (Planorbidae) induced oviposition in a high percentage of the experimental snails. Positive results of heterospecific injections between the first three species indicated a likely structural similarity of the ovulation hormones within the genus Lymnaea. The hormones of Lymnaea, Bulinus, and Biomphalaria, however, appeared to be different (negative results of heterogeneric injections).