Pollution of Polyaromatic Hydrocarbons in the Airborne Particles in the Developing Countries in Asia Region

Joint Research on Environmental Science and Technology for the Eart

Characterization of Luteinizing Hormone and Luteinizing Hormone Receptor and Their Indispensable Role in the Ovulatory Process of the Medaka

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

New evidence for the involvement of prostaglandin receptor EP4b in ovulation of the medaka, Oryzias latipes.

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 0h of ovulation. These results further substantiate that PGE(2)/Ptger4b signaling is involved in follicle rupture during ovulation in the medaka ovary

Expression and localization of collagen type IV α1 chain in medaka ovary

A cDNA clone coding for collagen type IV α1 chain was obtained from the ovary of the medaka, Oryzias latipes. The clone encodes a protein of 1639 amino acids including a putative 21-residue signal peptide, and the deduced amino acid sequence of the α1 chain was homologous to those of the proteins from other species. Collagen type IV α1 chain mRNA was expressed in various tissues of the adult fish. In situ hybridization analysis revealed that the α1 chain mRNA was localized in the follicle layer of all growing follicles. In the post-ovulatory follicle that had released its oocyte in ovulation, the α1 chain transcript was detected in a winding line surrounding the tissue. This localization pattern was different from that of gelatinase B, a marker gene for granulosa cells. A specific antibody was prepared for the medaka collagen type IV α1 chain. Immunohistochemical analysis using this antibody yielded results consistent with those obtained by the in situ hybridization experiment. The current results indicate that in the medaka ovary, collagen type IV is synthesized by theca cells and is localized in the basement membrane

The PGE2/Ptger4b pathway regulates ovulation by inducing intracellular actin cytoskeleton rearrangement via the Rho/Rock pathway in the granulosa cells of periovulatory follicles in the teleost medaka

We have previously shown that the prostaglandin E2/Ptger4b receptor system is involved in ovulation in teleost medaka and induces intracellular actin cytoskeleton rearrangement in the granulosa cells of preovulatory follicles. In this study, we investigated the signaling pathways through which prostaglandin E2 induces a change in the actin cytoskeleton. Treating preovulatory follicles with GW627368X (Ptger4b antagonist), a Rho inhibitor, or Y-27632 [Rho-associated protein kinase (Rock) inhibitor] inhibited not only in vitro follicle ovulation but also intracellular actin cytoskeleton rearrangement. Active Rhoa-c and Rock1 were detected in follicles immediately before ovulation. GW627368X also inhibited Rhoa-c activation and cytoskeleton rearrangement. PGE2-induced actin cytoskeleton rearrangement was not observed in the Ptger4b-, Rhoa-c-, or Rock1-deficient OLHNI-2 cells. These results indicate that the PGE2/Ptger4b pathway regulates intracellular actin cytoskeleton rearrangement via the Rho/Rock pathway in the granulosa cells of preovulatory follicles during medaka ovulation

Expression of cyclooxygenase-2 and prostaglandin receptor EP4b mRNA in the ovary of the medaka fish, Oryzias latipes: Possible involvement in ovulation

In vitro ovulation of mature medaka ovarian follicles was inhibited by inhibitors of cyclooxygenase (COX) or by an antagonist of the prostaglandin E2 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 E2 were also constant in the large-sized follicles during the spawning cycle. The expression of prostaglandin E2 receptor EP4b (ptger4b) mRNA was drastically upregulated in the large-sized follicles as the ovulation time approached. The current results indicate that prostaglandin E2. 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 E2 in the process

Amino acid sequence identity (%) between the medaka gonadotropin subunit and gonadotropin receptor proteins and those of other vertebrate species.

<p>Human (NP_001239312), mouse (NP_034019), chicken (XP_429886), <i>Xenopus</i> (NP_001085173), and zebrafish (NP_991250) for Gtha; human (NP_000501), mouse (NP_032071), chicken (NP_989588), <i>Xenopus</i> (NP_001084494), and zebrafish (NP_991187) for Fshb; human (NP_000885), mouse (NP_032523), chicken (ADY03193), <i>Xenopus</i> (NP_001079224), and zebrafish (AAV31153) for Lhb; human (NP_000136), mouse (NP_038551), chicken (NP_990410), <i>Xenopus</i> (NP_001243189), and zebrafish (AAP33512) for Fshra; and human (AAA59515), mouse (EDL38652), chicken (NP_990267), <i>Xenopus</i> (NP_001243190), and zebrafish (AAI62452) for Lhcgrbb.</p