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

Abstract

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 the12-amino acid sequence and 5' and 3' RACEs. These resulted in a 148 bp cDNA that coded a 43-amino acid sequence of H 2 N-VLSKQAHHHHHEGWSLPGVPAEIDDLAGNIDYNIFKEQREKIK-COOH. The synthetic 43-amino acid Aj-GSSL generated from this sequence induced GVBD in 50% of immature ovarian oocytes at 6 µM. 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 µM. This indicated that Aj-GSSL-P1 is of sufficient length for GSSL activity.

Spawning induced by cubifrin in the Japanese common sea cucumber Apostichopus japonicus

“…Katow et al [14] identified a gonad-stimulating substance-like peptide (GSSL) of around 4.6 kDa in the nerve tissue of A. japonicus that induced GVBD in vitro at a concentration of 30 lg/ml. The potential of GSSL to act as a spawning inducer was not examined in this study, but immunohistochemical observations suggested that holocrine secretion of GSSL-containing granules takes place into the body cavity.…”

One-step detection of matured females using immunochromatography measuring kit for aquaculture of the sea cucumber Apostichopus japonicus (Selenka)

“…The L-Ab and N-Ab were raised in mice using a synthetic peptide, AEIDDLAGNIDY, which constitutes a part of the physiologically active GSSL of A. japonicus (GenBank: Nucleotide AB434762) (Katow et al, 2009).…”

“…For immunoblotting analysis, protein aliquots were blotted to nitrocellulose membranes that were then incubated with alkaline phosphatase (AP)-tagged goat anti-mouse IgG Ab (Sigma-Aldrich, Inc., St. Louis, MO. USA), with the colour developed as described before (Katow et al, 2009).…”

“…Gonad-stimulating substance-like peptide (GSSL) is expressed during the breeding season of Apostichopus japonicus (Selenka) (Katow et al, 2009). Although GSSL is present in various organs, such as the body wall, warts, radial nerves, testes and ovaries, the gonads alone express the peptide in a manner closely associated with that of the breeding season (from March to June), whereas the other organs express GSSL throughout the year (Ahmed et al, 2011).…”

“…The present study aimed at developing a GSSL-immunochromatography measuring kit (GIM-Kit). Two antibodies [anti-GSSL-L antibody (L-Ab) and -N Ab (N-Ab)] were generated, and their specificity in the detection of the gonads in the matured stage has been previously observed (Katow et al, 2009;Ahmed et al, 2011). In this study, we report the successful creation and practical application of a GIM-Kit.…”