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First published online January 12, 2004
Journal of Experimental Biology 207, 621-632 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00785

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Purification and cDNA cloning of the ovigerous-hair stripping substance (OHSS) contained in the hatch water of an estuarine crab Sesarma haematocheir

Oleg Gusev¹, Hideki Ikeda¹, Tetsushi Okochi¹, Jae Min Lee², Masatsugu Hatakeyama², Chiyoko Kobayashi³, Kiyokazu Agata³, Hidenori Yamada⁴ and Masayuki Saigusa^1,*

¹ Laboratory of Animal Behavior and Evolution, Graduate School of Natural Science and Technology, Okayama University, Tsushima 3-1-1, Okayama 700-8530, Japan
² Developmental Mechanisms Laboratory, Developmental Biology Department, National Institute of Agrobiological Sciences, Owashi 1-2, Tsukuba 305-8634, Japan
³ Laboratory for Evolutionary Regeneration Biology, Center for Developmental Biology, RIKEN Kobe, Minatojima-minamimachi 2-2-3, Chuo-ku, Kobe 650-0047, Japan
⁴ Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima 3-1-1, Okayama 700-8530, Japan

^* Author for correspondence (e-mail: saigusa{at}cc.okayama-u.ac.jp)

Accepted 10 November 2003

The egg attachment system of an estuarine crab Sesarma haematocheir is formed on the maternal ovigerous hairs just after egg laying, and slips off these hairs just after hatching. The stripping is caused by an active factor that we call OHSS (ovigerous-hair stripping substance), which is released by the embryo upon hatching. OHSS was purified, and its active form had a molecular mass of 25 kDa. The cDNA of OHSS cloned from an embryonic cDNA library was 1759 bp long, encoding 492 amino acids in a single open reading frame (ORF). The C-terminal part of the predicted protein was composed of a trypsin-like serine protease domain, with homology to counterparts in other animals of 33–38%. The predicted protein (54.7 kDa) secreted as a zymogen may be cleaved post-translationally, separating the C-terminal from the N-terminal region. The OHSS gene was expressed in the embryo at least 2 weeks before hatching. Expression was also detected in the zoea larva 1 day after hatching and in the brain of the female. However, it was not detected in the muscle, hepatopancreas or ovigerous seta of the female. Ultrastructural analysis indicated that the material investing maternal ovigerous hair, i.e. the outermost layer (E1) of the egg case, is attached at the special sites (attachment sites) arranged at intervals of 130–160 nm on the hair. It is suggested that OHSS acts specifically at these sites, lysing the bond with the coat, thus disposing of the embryo attachment system. This enables the female to prepare the next clutch of embryos without ecdysis.

Key words: crab, Sesarma (or Chiromantes) haematocheir, ovigerous hair, embryo attachment system, investment coat, stripping, ovigerous-hair stripping substance (OHSS), serine protease

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