Gonad-stimulating substance-like molecule from the radial nerve of the sea cucumber

Gonad-stimulating substance-like molecule (GSSL) was isolated from the radial nerve of the sea cucumber, Apostichopus japonicus (Aj-GSSL), and its partial DNA and protein sequences were characterized. The smaller part of the molecule that also retains GSSL activity was estimated. Radial nerve extract (RNE) induced germinal vesicle breakdown (GVBD) at 3 mg/ml in 85% of immature ovarian oocytes. Similar intensity of GSSL activity to RNE was seen in a fraction that contained peptides between 3 kDa and 10 kDa (3-10 kDa-fraction) separated by ultrafiltlation membrane. MALDI-TOF MS analysis and silver-stained 18% SDS-PAGE slab gels identified a major peptide at around 4.6 kDa in a 3-10 kDa-fraction, and that was subjected to internal protein sequencing. The resulting 12-amino acid sequence was not found in the BLAST database to date. Immunohistochemistry using antiserum raised against the 12-amino acid peptide located the peptide to granular cells in the hyponeural part of the radial nerve and in the epineural sinus beneath the radial nerve. Sequence data was obtained using degenerate primers designed from the 12-amino acid sequence and 5 and 3 RACEs. These resulted in a 148 bp cDNA that coded a 43-amino acid sequence of H2N-VLSKQAHHHHHEGWSLPGVPAEIDDLAGNIDYNIFKEQREKIK-COOH. The synthetic 43-amino acid Aj-GSSL generated from this sequence induced GVBD in 50% of immature ovarian oocytes at 6 microM. An N-terminal 21-amino acid peptide of the synthetic partial Aj-GSSL (Aj-GSSL-P1) induced GVBD to 80% of immature ovarian oocytes at 12 microM. This indicated that Aj-GSSL-P1 is of sufficient length for GSSL activity.