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 as a model, we found that in vitro follicle ovulation, but not germinal vesicle breakdown, was inhibited by three gap junction blockers: carbenoxolone, mefloquine, and flufenamic acid. These blockers specifically reduced the follicular expression of matrix metalloproteinase-15 (mmp15) mRNA and protein (Mmp15), a protease essential for medaka ovulation. This suggests that gap junctional communication is critical for successful ovulation and mmp15/Mmp15 expression.

Further experiments with carbenoxolone, chosen as a representative blocker, revealed that while the expression of nuclear progestin receptor (Pgr)—a transcription factor required for mmp15 expression—was unaffected, the formation of phosphorylated Pgr was significantly suppressed. Carbenoxolone treatment also reduced Pgr binding to the mmp15 promoter region. Additionally, mRNA expression of cyclin-dependent protein kinase-9 (cdk9) and cyclin I (ccni), whose protein products are involved in Pgr phosphorylation in ovulating follicles, was suppressed by carbenoxolone.

Transcripts of connexin 34.5 (cx34.5) and connexin 35.4 (cx35.4) were AZD4573 predominantly expressed in the follicle cells of ovulating follicles. These findings highlight the critical role of gap junctional communication in medaka ovulation.