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

Abstract

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: adam8b (a disintegrin and metalloproteinase domain 8b), adamts8a (a disintegrin and metalloproteinase with thrombospondin motif 8a), adamts9, mmp2 (matrix metalloproteinase 2), and mmp9 in the nuclear progestin receptor knockout (pgr−/−) 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 adamts1 in preovulatory follicles at different times within the spawning cycle in pgr−/− and wildtype (wt) zebrafish and under varying hormonal treatments. We found that transcripts of adam8b, adamts1, adamts9, and mmp9 increased drastically in the preovulatory follicular cells of wt female zebrafish, while changes of adamts8a and mmp2 were not significant. This increase of adam8b, adamts9, and mmp9 was significantly reduced in pgr−/−, whereas expression of adamts1 was not affected in pgr−/− zebrafish. Among upregulated metalloproteinases, adamts9 mRNA was found to be expressed specifically in follicular cells. Strong immunostaining of Adamts9 protein was observed in the follicular cells of wt fish, and this expression was reduced drastically in pgr−/−. Interestingly, about an hour prior to the increase of metalloproteinases in wt fish, both Pgr transcript and protein increased transiently in preovulatory follicular cells. The results from in vitro experiments showed that adamts9 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 adamts9 occurs specifically in preovulatory follicular cells of zebrafish prior to ovulation. Progestin and its receptor (Pgr) are essential for the upregulation of metalloproteinases.

Downregulation of nuclear progestin receptor (Pgr) and subfertility in double knockouts of progestin receptor membrane component 1 (pgrmc1) and pgrmc2 in zebrafish

“…The role of Pgr in ovulation is highly conserved across vertebrate species, as is evidenced by the inability of Pgr knockout female zebrafish, rats, and mice to ovulate (Kubota et al 2016, Lydon et al 1995, Zhu et al 2015. Further studies indicated that Pgr is an upstream regulator important for the increased expression of proteolytic enzymes and therefore promotes the rupture of follicles for releasing mature oocytes during ovulation (Liu et al, 2017(Liu et al, , 2018.…”

GFP expression pattern in pituitary and gonads under the control of nuclear progesterone receptor promoter in transgenic zebrafish

“…46 Our previous study identified adamts9, expressed in follicle cells, as a downstream target of Pgr, which is essential for ovulation in zebrafish. 47,48 However, it is worth noting that theca cells are also vital to the mammalian ovulatory process. 49 It has been reported that Adamts1 mRNA in the theca interna was upregulated just prior to ovulation in the horse.…”

“…Since PGR is expressed by granulosa cells and plays a critical role in ovulation, studies have focused on granulosa cells and revealed that PGR‐regulated genes unequivocally linked to the ovulatory success are the extracellular matrix proteases, for example, ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family members 46 . Our previous study identified adamts9 , expressed in follicle cells, as a downstream target of Pgr, which is essential for ovulation in zebrafish 47,48 . However, it is worth noting that theca cells are also vital to the mammalian ovulatory process 49 .…”

Delay in Primordial Germ Cell Migration in Adamts9 Knockout Zebrafish

“…Various stages of AB wildtype embryos were collected at different times of development. Ovarian follicles were divided into different stages according to follicular size and morphological criteria [26] with a slight modi cation [21][22][23] . Stage I, III, IVb and V ovarian follicles were collected from 4-month old mature female AB wildtype sh between 7:00am and 8:30am (lights on photoperiod 8:30am-10:30pm).…”

“…Our previous studies suggest Adamts9 is critical for normal development of ovaries and ovulation in the zebra sh [21][22][23][24] . In the present study, we examined expression of Adamts9 in the embryonic development and its roles in germ cell migration.…”