ts of a 19320832 representative experiment are presented. Preovulatory follicles isolated from spawning medaka ovaries 22 h before ovulation were treated with or without PMSG. Collection of follicles, total RNA extraction, and real-time RT-PCR of mmp15 were conducted as in. The expression levels were normalized to that of actb and expressed as the fold change compared to the levels of the 222 h follicle. The results are presented as the mean 6S.E.M.PMSG induced in vitro ovulation of the preovulatory follicles. In this context, we should note the strictness of ligand-binding specificity of medaka Lhcgrbb because limited hormone-binding selectivity is a distinct feature of teleost gonadotropin bioactivity. We found that treatment of the medaka Fshraexpressing HEK 293T cells with medaka rLh resulted in a slight increase in the luciferase activity. This finding might indicate a cross-activation of Fshra, that is, an interaction of Fshra with Lh in the medaka. To establish the strict specificities of medaka gonadotropin receptors, further studies are required. Nevertheless, the medaka gonadotropin receptors appear to be specific for their cognate gonadotropins compared with those from other teleost species. For example, zebrafish Fshra responds to goldfish pituitary extract and bovine FSH, while Lhcgrbb of the same fish can be activated by the pituitary extract, hCG, bovine FSH and bovine LH. More recently, specificities of eel, trout, and tilapia gonadotropin receptors for various gonadotropins have been compared. Generally, the results of that comparative study seem to support the broad specificity 
of fish gonadotropin receptors. In the present study, we showed that rLh was a gonadotropin capable of inducing ovulation in vitro by postvitellogenic follicles in medaka. In addition, the follicles incubated with rLh synthesized Mmp15, an MMP indispensable for follicle rupture during ovulation in the fish. These results indicate that Lh is involved in fish ovulation. Our data also indicate that the preovulatory follicles that are destined to ovulate undergo an Lh surge between 19 and 15 h before ovulation in vivo. It should be noted that we failed to produce medaka recombinant Fsh in the present study. This did not allow us to test the potential binding of medaka Fsh to medaka 22860184 Lhcgrbb. Therefore, we cannot exclude completely the possibility that Fsh plays a role in medaka ovulation. It is worthwhile to consider the relevance and 169939-93-9 usefulness of the in vitro rLh-supplemented follicle culture system reported in this study. To date, many in vitro methods using ovary fragments and ovarian follicles have been elaborated for various teleost species. They include zebrafish, Atlantic croaker, rainbow trout, brook trout, goldfish, sea lamprey, coho salmon, European sea bass, and killifish. Although these experimental models generally are good systems for studying oocyte maturation of the respective teleost species, mature, healthy and intact oocytes cannot come off the follicle or ovarian fragments even when they have been primed by gonadotropins in vivo. To our knowledge, the current rLhsupplemented culture method using medaka preovulatory follicles in vitro is the only experimental system that allows the follicles to undergo oocyte maturation as well as ovulation at rates as efficient as in vivo. PMSG has an Lh-like activity on medaka preovulatory follicles. Our present experiments using HEK 293T cells expressing medaka Lhcgrbb confirmed the binding of PMSG to L