A cDNA for a prostaglandin E(2) (PGE(2)) receptor subtype 4, EP4b (Ptger4b), was cloned from the medaka ovary. The effect of PGE(2) was examined using COS-7 cells expressing the recombinant Ptger4b protein. An increase in intracellular cAMP levels was observed when the cells were incubated with PGE(2), but the increase in cAMP levels was nullified by the addition of the EP4 antagonist GW627368X. The expression of ptger4b mRNA was drastically induced by the addition of pregnant mare serum gonadotropin to the in vitro culture of large preovulatory follicles. In in vitro ovulation studies of the effect of GW627368X addition on follicle ovulation, the critical timing of the PGE(2)/Ptger4b interaction was suggested to be between -1 and 0 h of ovulation. These results further substantiate that PGE(2)/Ptger4b signaling is involved in follicle rupture during ovulation in the medaka ovary.

Ovulation denotes the discharge of fertilizable oocytes from ovarian follicles. Follicle rupture during ovulation requires extracellular matrix (ECM) degradation at the apex of the follicle. In the teleost medaka, an excellent model for vertebrate ovulation studies, LH-inducible matrix metalloproteinase 15 (Mmp15) plays a critical role during rupture. In this study, we found that follicle ovulation was inhibited not only by roscovitine, the cyclin-dependent protein kinase (CDK) inhibitor, but also by CDK9-inhibitor II, a specific CDK9 inhibitor. Inhibition of follicle ovulation by the inhibitors was accompanied by the suppression of Mmp15 expression in the follicle. In follicles treated with the inhibitors, the formation of the phosphorylated nuclear progestin receptor (Pgr) was inhibited. Roscovitine treatment caused a reduction in the binding of Pgr to the promoter region of mmp15. The expression of Cdk9 and cyclin I (Ccni), and their association in the follicle was demonstrated, suggesting that Cdk9 and Ccni may be involved in the phosphorylation of Pgr in vivo. LH-induced follicular expression of ccni/Ccni was also shown. This study is the first to report the involvement of CDK in ECM degradation during ovulation in a vertebrate species.

In vitro ovulation of mature medaka ovarian follicles was inhibited by inhibitors of cyclooxygenase (COX) or by an antagonist of the prostaglandin E(2) receptor (EP). Of the three medaka COX genes, ptgs2 was most dominantly expressed in the fish ovary. The ptgs2 transcript was detected in all sizes of growing follicles. In a 24-h spawning cycle, large-sized follicles contained ptgs2 mRNA at a fairly constant level. The levels of COX enzyme activity and prostaglandin E(2) were also constant in the large-sized follicles during the spawning cycle. The expression of prostaglandin E(2) receptor EP4b (ptger4b) mRNA was drastically upregulated in the large-sized follicles as the ovulation time approached. The current results indicate that prostaglandin E(2), which might be produced by COX-2, is involved in the ovulation of medaka, and that EP4b is likely the receptor responsible for exerting the action of prostaglandin E(2) in the process.

Using medaka, we found that in vitro follicle ovulation, but not germinal vesicle breakdown, was inhibited by three gap junction blockers, carbenoxolone, mefloquine, and flufenamic acid. The blockers specifically inhibited follicular expression of matrix metalloproteinase-15 mRNA and the protein (mmp15/Mmp15), a protease indispensable for medaka ovulation, indicating that gap junctional communication may be required for successful ovulation and mmp15/Mmp15 expression. Further experiments using carbenoxolone as the representative of the gap junction blockers showed that expression of nuclear progestin receptor (Pgr), a transcription factor required for mmp15 expression, was not affected by carbenoxolone treatment, but the formation of phosphorylated Pgr was considerably suppressed. Carbenoxolone treatment caused a decrease in the Pgr binding to the promoter region of mmp15. mRNA expression of cyclin-dependent protein kinase-9 (cdk9) and cyclin I (ccni), whose translation products are demonstrated to be involved in Pgr phosphorylation in the medaka ovulating follicles, was suppressed by carbenoxolone treatment. Transcripts of connexin 34.5 (cx34.5) and connexin 35.4 (cx35.4) were dominantly expressed in the follicle cells of ovulating follicles. The results indicate that gap junctional communication plays an important role in medaka ovulation.

Hormonal regulation of the expression of Mmp15, a proteolytic enzyme indispensable for ovulation in the teleost medaka, was investigated. In an in vitro culture system using preovulatory follicles, Mmp15 expression and ovulation were induced in the presence of recombinant luteinizing hormone (rLh). Both rLh-induced Mmp15 expression and ovulation were 17α, 20β-dihydroxy-4-pregnen-3-one-dependent, suggesting the involvement of a nuclear progestin receptor (Pgr). In vitro follicle ovulation and Mmp15 expression were reduced by treatment with the Pgr antagonist RU-486. Like Pgr, the transcription factor CCAAT/enhancer-binding protein β (Cebpb) was induced by rLh. ChIP analyses indicated that Pgr and Cebpb bound to the mmp15 promoter region. These results indicate that the rLh-induced expression of Mmp15 is mediated by Pgr and Cebpb. A differential timing of expression of Pgr and Cebpb in the preovulatory follicles appears to explain the considerably long time-lag from the pgr gene activation to mmp15 gene expression.

The molecular properties and roles of luteinizing hormone (Lh) and its receptor (Lhcgrbb) have not been studied for the medaka (Oryzias latipes), which is an excellent animal model for ovulation studies. Here, we characterized the medaka Lh/Lhcgrbb system, with attention to its involvement in the ovulatory process of this teleost fish. In the medaka ovary, follicle-stimulating hormone receptor mRNA was expressed in small and medium-sized follicles, while lhcgrbb mRNA was expressed in the follicle layers of all growing follicles. Experiments using HEK 293T cells expressing medaka Lhcgrbb in vitro revealed that gonadotropin from pregnant mare's serum and medaka recombinant Lh (rLh) bound to the fish Lhcgrbb. The fish gonadotropin subunits Gtha, Fshb, and Lhb were essentially expressed at fairly constant levels in the pituitary of the fish during a 24-h spawning cycle. Using medaka rLh, we developed a follicle culture system that allowed us to follow the whole process of oocyte maturation and ovulation in vitro. This follicle culture method enabled us to determine that the Lh surge for the preovulatory follicle occurred in vivo between 19 and 15 h before ovulation. The present study also showed that oocyte maturation and ovulation were delayed several hours in vitro compared with in vivo. Treatment of large follicles with medaka rLh in vitro significantly increased the expression of Mmp15, which was previously demonstrated to be crucial for ovulation in the fish. These findings demonstrate that Lh/Lhcgrbb is critically involved in the induction of oocyte maturation and ovulation.