Gonadotropins (GTHs), FSH and LH, play central roles in vertebrate reproduction. Here, we report the production of biologically-active recombinant FSH (r-mtFSH) and LH (r-mtLH) of an endangered salmon species, Manchurian trout (Brachymystax lenok), by baculovirus in silkworm (Bombyx mori) larvae. The biological activities of the recombinant hormones were analyzed using COS-7 cell line transiently expressing either amago salmon FSH or LH receptor. The steroidogenic potency of the r-mtFSH and r-mtLH was examined by a culture system using rainbow trout follicles in vitro. In vivo, bioactivity was assessed by measuring ovarian weight, oocyte diameter, and plasma steroid hormone levels in female rainbow trout. Moreover, inducing potency of milt production were examined in vivo using goldfish. Our results demonstrated that the r-mtFSH and r-mtLH were successfully produced in the baculovirus-silkworm system and recognized by their cognate receptors specifically in vitro. The production of estradiol-17b (E2) and testosterone (T) was stimulated by the r-mtFSH and r-mtLH respectively, from the full-grown follicles of rainbow trout, whereas both E2 and T were increased by relatively higher doses of the recombinant hormones from the follicles of the maturing stage. In in vivo assay, injection of the r-mtFSH but not r-mtLH increased ovarian weight, oocyte diameter, and plasma E2 levels in immature rainbow trout. Injection of both r-mtFSH and r-mtLH induced milt production in male goldfish. In conclusion, the present study strongly suggests that the r-mtFSH and r-mtLH have distinct biological properties, such as a specific responsiveness for the cognate receptor, steroidogenic, and vitellogenic activities for ovarian follicles in salmonids. These recombinant FSH and LH may be applied for future studies on the gonadal development and maturation in fishes as well as the endangered salmon species.

Manchurian trout Brachymystax lenok is an endangered fish species in East Asia including the Korean peninsula. To establish a method for artificial propagation and to improve our understanding of reproduction in the species, cloning of the pituitary gonadotropin (GTH) subunit cDNAs encoding the Manchurian trout gonadotropin common α (mtGTHα), follicle-stimulating hormone β (mtFSHβ), and luteinizing hormone β (mtLHβ) by polymerase chain reaction, were attempted. The open reading frames of the mtGTHα, mtFSHβ, and mtLHβ encoded 114, 137, and 142 amino acid residues, respectively. The mature peptides showed strikingly high sequence identities to those of salmonid species GTHα2, FSHβ, and LHβ subunits (92-100%). In an examination of tissue distribution of the mtGTHα, mtFSHβ, and mtLHβ, it was revealed that all of the three subunit genes are specifically expressed in the pituitary by reverse transcription-polymerase chain reaction analysis. Next, single-chain recombinant FSH and LH constructs using the mtGTHα, mtFSHβ, and mtLHβ cDNAs were produced. In a transient transfection of the FSH and LH constructs into Chinese hamster ovary (CHO-K1) cells followed by Western blot analysis, the tethered FSH in cell lysates or LH secreted into a medium was detected from 48 to 72 h after transfection. The present study provides a possibility of bioactive GTH production for studies on the reproductive physiology of GTH in the Manchurian trout.