Production of recombinant channel catfish (Ictalurus punctatus) FSH and LH in S2 Drosophila cell line and an indication of their different actions

Abstract

Due to the lack of purified, native gonadotropins (GtH) for almost all species of fish, we designed a system for the production of recombinant bioactive luteinizing hormone (LH) and follicle stimulating hormone (FSH) using the channel catfish (Ictalurus punctatus) as a model animal. The strategy was to produce the three subunits composing FSH and LH, i.e. the common a-subunit (a-glycoprotein hormone (a-GP)), b-FSH, and b-LH subunit, individually in stable recombinant insect cells (S2) with C-terminal His-tag. This expression system was also used to co-express the a-subunit without the His-tag with each of the His-tagged b-subunits. The recombinant S2 cells were capable of secreting FSH and LH heterodimers and a-GP in abundance; however, expression of the individual b-subunits was much less successful. The recombinant GtHs were partially purified from the cell medium by immobilized metal affinity chromatography to w15% purity with a yield of 7 and 4 mg per liter of medium for FSH and LH respectively. These recombinant GtHs activated their receptors in vitro, enhanced estrogen secretion, up-regulated several steroidogenic enzyme genes in channel catfish ovarian follicles, and increased androgen secretion from African catfish testis. Interestingly, the FSH and LH dose-response curves for each of these biological activities clearly demonstrate differences in their cellular action and physiological roles. This expression system may be an important development for the production of speciesspecific GtHs so that FSH-and LH-specific mechanisms of actions within the reproductive endocrine processes can finally be examined with homologous, albeit recombinant, hormones.

Sertoli cell proliferation in the adult testis is induced by unilateral gonadectomy in African catfish

“…Irrespective of the mechanism(s) involved, our This has been shown in Japanese eel [31], African catfish [17], and zebrafish [18]. Indeed, Fsh was 23 more potent as steroidogenic hormone than Lh in both catfish [60] and zebrafish [18]. Moreover, Fsh…”

Effects of reproductive stage, GH, and 11-ketotestosterone on expression of growth differentiation factor-9 in the ovary of the eel, Anguilla australis

“…Terminal sampling took place on day 12 during which BW was recorded and gonad tissue recovered and snap frozen until analysis for routine histology and gdf9 mRNA levels. Recombinant jeGH-I and -II (Kishida et al 1987) were produced using the Drosophila S2 expression system established for production of piscine gonadotropins (Zmora et al 2007, Kazeto et al 2008. Fine details of the GH expression and isolation procedures are to follow (Y Kazeto, S Akiyama, Y Ozaki, T Todo, S Adachi & K Yamauchi, unpublished observations).…”

Expression of recombinant zebrafish follicle-stimulating hormone (FSH) in methylotropic yeast Pichia pastoris

“…One of the approaches taken to solve this problem has been to produce fish gonadotropins using different bioreactors (Aizen et al 2007;Fidler et al 2003;Kamei et al 2003;Ko et al 2007;Kobayashi et al 2006;So et al 2005;Vischer et al 2003;Zmora et al 2007). The production of both the native and fusion forms of gonadotropins have been reported using this approach; however, to date no study has compared the bioactivity of the two forms of gonadotropins produced using the same platform of production.…”

“…One of the hurdles to the study of gonadotropin functions in fish is the lack of hormones, especially the homologous ones from the same species. To solve this problem, several laboratories, including ours, have recently reported the production of recombinant fish gonadotropins using various expression systems as the bioreactors, including the soil amoeba Dictyostelium discoideum (Vischer et al 2003), insect cells (Zmora et al 2007), yeast Pichia pastoris (Aizen et al 2007;Kamei et al 2003;Kasuto and Levavi-Sivan 2005), baculovirus (Cui et al 2007;Hayakawa et al 2008;Ko et al 2007) and mammalian cells .…”