Biochemical and immunohistochemical studies of the egg-laying hormone of Aplysia californica : purification, primary structure, neurosecretion and morphological distribution.

Abstract

Egg-laying behavior in Aplysia californica can be triggered by the introduction of a neuropeptide, the Egg-Laying Hormone (ELH) into the circulation. ELH is synthesized by the neurosecretory bag cells of the abdominal ganglion and released when these neurons are induced to fire repetitively. In this thesis, biochemical and immunohistoehemical techniques have been employed to study the primary structure, release and distribution of ELH in the nervous system of Aplysia. The purification of ELH to homogeneity from extracts of bag cell clusters, and the analysis of its primary structure are discussed in Chapter 1. A 100-fold enrichment of bioactive material was obtained by cation exchange chromatography (Sephadex SP C25) followed by gel filtration (BioRad P-6). This purified material was determined to be homogeneous by four lines of analysis: (i) SDS polyacrylamide gel electrophoresis, (ii) isoelectric focussing, (iii) microsequence analysis, and (iv) comparison of the amino acid compositions from acid hydrolysis and from microsequence data. ELH is a 36 amino acid, basic peptide with a calculated molecular weight of 4385 and an apparent isoelectric point of 9.0-9.2. Its amino acid sequence was determined as: H-lle-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu-Gln-lleArg-Glu-Arg-Gln-Arg-Tyr-Leu-Ala-Asp-Leu-Arg-Gln-Arg-Leu-Leu-Glu-Lys-OH Chapter 2 demonstrates the release of ELH, identified by molecular weight, pI and bioactivity, when bag cell clusters afterdischarge in vitro. During such synchronous and prolonged electrical activity, bag cell clusters, which have been pulsed in 35S-Met, secrete at least four labeled presumed peptides of different molecular weights. One of these comigrates with 3H-Leu labeled, purified ELH on gel filtration chromatography and causes egg laying when injected into a test animal. This material also comigrates with 3H-ELH on isoelectrifocussing gels. A second released peptide has a molecular weight of approximately 5-6 K and a pI of 4.8; its function, and the functions of the other released molecules, are unknown. In order to study the distribution of ELH in the nervous system of Aplysia, antibodies were generated against the purified neuropeptide, coupled to a carrier molecule, thyroglobulin (Tg). Immune sera, enriched for anti-ELH antibodies by passage through an affinity column to remove antibodies which bound to Tg, was used for localizing ELH-like immunoreactivity in frozen sections of Aplysia ganglia. These results were discussed in Chapters 3 and 4. When sections of the abdominal ganglion were stained by the PAP method for ELH, all neurons within the bag cell clusters were found to be immunoreactive. Except for occasionally displaced bag cells, all neurons within the ganglion remained unreactive, reflecting the specificity of the antiserum. Immunopositive processes from bag cells proliferate in the vascularized connective tissue capsule which serves as a neurohemal organ facilitating release of neurohormones. Some processes form a spiralling cuff around the nerve trunks of the pleuro-visceral connective and the vulvar nerves; others invade the ganglion in association with connective tissue septa which form partitions between groups of neurons. Immunoreactive fibers with varicosities are also found within the neuropile and the commissure between the two hemiganglia. This light microscopic visualization of the bag cell neuroendocrine system provides morphological support for the model of local hormone action of ELH upon other neurons in the abdominal ganglion. The immunoreactivity of all neurons within the clusters provides the strongest evidence to date of the homogeneity of the bag cell population. Antibodies generated against ELH from A. californica selectively stained the bag cell systems of three other species of Aplysia - A. braziliana, A. vaccaria and A. dactylomela - which also share cross bioactivity. It is likely that receptor binding sites and antigenic determinants are conserved in their ELHs. The fourth chapter describes the organization of cells and fiber tracts with ELH-like immunoreactivity, endogenous to the head ganglia. Each pleural ganglion has 1-5 immunopositive somata which are strikingly similar to bag cells in cell and nuclear sizes, process morphology and location. These similarities, coupled with the close developmental association of the pleural and abdominal ganglia, suggest a common heritage for both populations of ELH+ cells. The ELH immunoreactive system in the cerebral ganglion consists of two laterally located clusters of small cells on the dorsal surface of the ganglion and extensive fiber tracts throughout the neuropile. The nature of immunoreactive molecules and the function of these systems within the cerebral and pleural ganglia are unknown. However, perfusion of ELH is known to induce long-term changes in the electrical activity of head ganglia neurons in vitro, and some of these changes may be linked to the suppression of feeding and locomotion during egg laying. The presence of these immunopositive systems in the pleural and cerebral ganglia raises the possibility that ELH target neurons in head ganglia may respond to local sources of ELH or ELH-like molecules.