Inhibition of medaka ovulation by gap junction blockers due to its disrupting effect on the transcriptional process of LH-induced Mmp15 expression

Abstract

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.