Progestin and Nuclear Progestin Receptor Are Essential for Upregulation of Metalloproteinase in Zebrafish Preovulatory Follicles.

Ovulation requires proteinases to promote the rupture of ovarian follicles. However, the identity of these proteinases remains unclear. In our previous studies using RNA-seq analysis of differential expressed genes, we found significant down-regulation of five metalloproteinases: (a disintegrin and metalloproteinase domain 8b), (a disintegrin and metalloproteinase with thrombospondin motif 8a), (matrix metalloproteinase 2), and in the nuclear progestin receptor knockout ( ) zebrafish that have failed to ovulate. We hypothesize that these metalloproteinases are responsible for ovulation and are regulated by progestin and Pgr. In this study, we first determined the expression of these five metalloproteinases and in preovulatory follicles at different times within the spawning cycle in and wildtype () zebrafish and under varying hormonal treatments. We found that transcripts of , and increased drastically in the preovulatory follicular cells of female zebrafish, while changes of and were not significant. This increase of , and was significantly reduced in , whereas expression of was not affected in zebrafish. Among upregulated metalloproteinases, mRNA was found to be expressed specifically in follicular cells. Strong immunostaining of Adamts9 protein was observed in the follicular cells of fish, and this expression was reduced drastically in . Interestingly, about an hour prior to the increase of metalloproteinases in fish, both Pgr transcript and protein increased transiently in preovulatory follicular cells. The results from experiments showed that expression markedly increased in a dose, time and Pgr-dependent manner when preovulatory follicles were exposed to a progestin, 17α,20β-dihydroxy-4-pregnen-3-one (DHP). Taken together, our results provide the first evidence that upregulation of occurs specifically in preovulatory follicular cells of zebrafish prior to ovulation. Progestin and its receptor (Pgr) are essential for the upregulation of metalloproteinases.