The maturation and ovulation of fish oocytes are well-characterized biological processes induced by progestins via coordination of nongenomic actions and genomic actions. Previously, we established a procedure that enables the induction of oocyte maturation and ovulation in live zebrafish by simple administration of the natural teleost maturation-inducing hormone 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-DHP) into the surrounding water. By this in vivo assay, the potencies of chemicals in inducing or preventing oocyte maturation and ovulation can be evaluated. The potencies of compounds in inducing ovulation of zebrafish oocytes also can be evaluated in vivo with improved in vitro assays. Here, we attempted to evaluate the effect of Org OD 02-0 (Org OD 02), a selective agonist for membrane progestin receptor (mPR), on fish oocyte maturation and ovulation with in vitro and in vivo assays. As reported previously, Org OD 02 triggered oocyte maturation in vitro. The same Org OD 02 triggered oocyte maturation within several hours in vivo. Surprisingly, Org OD 02 even induced ovulation both in in vivo and in vitro. Eggs from Org OD 02-induced ovulation could be fertilized by artificial insemination. The juveniles developed normally. These results indicated that Org OD 02 triggered physiological ovulation in live zebrafish. In summary, we have demonstrated the effect of Org OD 02 on fish oocyte maturation and ovulation in vitro and in vivo. The results suggested that Org OD 02 acted as an agonist not only of mPR but also of nuclear progesterone receptor (nPR). (C) 2020 Elsevier Inc. All rights reserved.