Progestins, progesterone derivatives, are the most critical signaling steroid for initiating final oocyte maturation (FOM) and ovulation, in order to advance fully-grown immature oocytes to become fertilizable eggs in basal vertebrates. It is well-established that progestin induces FOM at least partly through a membrane receptor and a non-genomic steroid signaling process, which precedes progestin triggered ovulation that is mediated through a nuclear progestin receptor (Pgr) and genomic signaling pathway. To determine whether Pgr plays a role in a non-genomic signaling mechanism during FOM, we knocked out Pgr in zebrafish using transcription activator-like effector nucleases (TALENs) and studied the oocyte maturation phenotypes of Pgr knockouts (Pgr-KOs). Three TALENs-induced mutant lines with different frame shift mutations were generated. Homozygous Pgr-KO female fish were all infertile while no fertility effects were evident in homozygous Pgr-KO males. Oocytes developed and underwent FOM normally in vivo in homozygous Pgr-KO female compared to the wild-type controls, but these mature oocytes were trapped within the follicular cells and failed to ovulate from the ovaries. These oocytes also underwent normal germinal vesicle breakdown (GVBD) and FOM in vitro, but failed to ovulate even after treatment with human chronic gonadotropin (HCG) or progestin (17α,20β-dihydroxyprogesterone or DHP), which typically induce FOM and ovulation in wild-type oocytes. The results indicate that anovulation and infertility in homozygous Pgr-KO female fish was, at least in part, due to a lack of functional Pgr-mediated genomic progestin signaling in the follicular cells adjacent to the oocytes. Our study of Pgr-KO supports previous results that demonstrate a role for Pgr in steroid-dependent genomic signaling pathways leading to ovulation, and the first convincing evidence that Pgr is not essential for initiating non-genomic progestin signaling and triggering of meiosis resumption.

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Our previous study showed that the in vivo positive effects of 17α,20β-dihydroxy-4-pregnen-3-one (DHP), the major progestin in zebrafish, on early spermatogenesis was much stronger than the ex vivo ones, which may suggest an effect of DHP on the expression of gonadotropins. In our present study, we first observed that fshb and lhb mRNA levels in the pituitary of male adult zebrafish were greatly inhibited by 3 wk exposure to 10 nM estradiol (E2). However, an additional 24 hr 100 nM DHP exposure not only reversed the E2-induced inhibition, but also significantly increased the expression of fshb and lhb mRNA. These stimulatory effects were also observed in male adult fish without E2 pretreatment, and a time course experiment showed that it took 24 hr for fshb and 12 hr for lhb to respond significantly. Because these stimulatory activities were partially antagonized by a nuclear progesterone receptor (Pgr) antagonist mifepristone, we generated a Pgr-knock out (pgr−/−) model using the TALEN technique. With and without DHP in vivo treatment, fshb and lhb mRNA levels of pgr−/− were significantly lower than those of pgr+/+. Furthermore, ex vivo treatment of pituitary fragments of pgr−/− with DHP stimulated lhb, but not fshb mRNA expression. Results from double-colored fluorescent in situ hybridization showed that pgr mRNA was expressed only in fshb-expressing cells. Taken together, our results indicated that DHP participated in the regulation of neuroendocrine control of reproduction in male zebrafish, and exerted a Pgr-mediated direct stimulatory effect on fshb mRNA at pituitary level.

14Recently, we found anovulation in nuclear progestin receptor (Pgr) knockout (Pgr-KO) zebrafish, 15 which offers a new model for examining Pgr regulated genes and pathways that are important for 16 ovulation and fertility. In this study, we examined expression of all transcripts using RNA-Seq in 17 pre-ovulatory follicular cells collected after the final oocyte maturation, but prior to ovulation, 18 from wild-type (WT) or Pgr-KO fish. Differential expression analysis revealed 2,888 genes 19 significantly differentially expressed between WT and Pgr-KO fish. Among those, 1,230 gene 20 transcripts were significantly more expressed, while 1,658 genes were significantly less expressed 21 in WT than those in Pgr-KO. We then retrieved and compared transcriptional data from online 22 databases and further identified 661 conserved genes in fish, mice, and humans, that showed 23 similar levels of high (283 genes) or low (387) expression in animals that were ovulating compared 24 to those with no ovulation. For the first time, ovulatory genes and their involved biological 25 processes and pathways were also visualized using Enrichment Map and Cytoscape. Intriguingly, 26 enrichment analysis indicated the genes with higher expression were involved in multiple 27 ovulatory pathways and processes such as inflammatory response, angiogenesis, cytokine 28 production, cell migration, chemotaxis, MAPK, focal adhesion, and cytoskeleton reorganization. 29In contrast, the genes with lower expression were mainly involved DNA replication, DNA repair, 30DNA methylation, RNA processing, telomere maintenance, spindle assembling, nuclear acid 31 transport, catabolic processes, nuclear and cell division. Our results indicate that a large set of 32 genes (>3,000) are differentially regulated in the follicular cells in zebrafish prior to ovulation, 33 terminating programs including growth and proliferation, and beginning processes including the 34 inflammatory response and apoptosis. Further studies are required to establish relationships among 35 these genes and an ovulatory circuit in zebrafish model. 37Ovulation is a physiological process that releases a fertilizable oocyte from follicular cells and is 38 an essential reproductive event for the preservation of a species. It is well established that 39 luteinizing hormone (LH) initiates a cascade of signaling; including upregulation of progestin and 40 its nuclear progestin receptor (PGR) which activates various downstream targets and signaling 41 pathways, eventually leading to follicular rupture. However, our understanding of the molecular 42 mechanisms that control ovulation is far from complete. For example, there is limited evidence of 43 downstream targets and signaling pathways that PGR regulates. A few genome-wide transcriptome 44 analyses of differentially expressed genes in the follicular cells of pre-ovulatory oocytes suggest 45 conserved gene expression regulation in humans, macaques, and mice [1-3]. To our knowledge, 46 there are no published transcriptomic analyses of gene e...