Abstract
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.
Highlights
Steroid hormone-regulated physiological processes mediated by steroid hormone receptors that act as transcriptional factors and co-regulators in the nucleus, i.e., genomic steroid signaling pathway, have been well-established
GENERATION OF progestin receptor (Pgr)-KO ZEBRAFISH MODEL Based on the analyses of restriction enzyme digestion and DNA sequence of genomic DNA extracted from a pool of 30 embryos at 2dpf microinjected with transcription activator-like effector nucleases (TALENs) mRNAs, both pairs of TALENs effectively induce insertion/deletion mutations
TALENs directed toward target site 2 generated a 10 nucleotide deletion (TGGATGTCCG, nucleotide positions 616–625, Figures 2 and 4) that resulted in truncation of amino acids 263–617 and alteration of amino acids 206–263 in the coding sequence (Figure 4)
Summary
Steroid hormone-regulated physiological processes mediated by steroid hormone receptors that act as transcriptional factors and co-regulators in the nucleus, i.e., genomic steroid signaling pathway, have been well-established. Both previously established nuclear steroid receptors and new classes of proteins have been suggested to be the candidate receptor(s) for non-genomic steroid signaling processes [4,5,6,7,8,9]. The role of these receptors as mediators of non-genomic steroid signaling has been vigorously debated but relatively few explicit tests of their function have been conducted. Further research is required to determine whether these proposed receptors act as non-genomic steroid receptor(s) in specific physiological process
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call