Supplement: To Characterize Nongenomic Progestin Receptors via Knockouts in Zebrafish

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Ovulation is an indispensable reproductive event; yet our understanding of the molecular mechanisms that control ovulation is still incomplete. Activation of proteases leading to extracellular matrix disassociation is an essential step for follicle rupture and ovulation to proceed. Involvement of several metalloproteinase families including members of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), MMPs (matrix metalloproteinases), and ADAM (a disintegrin and metalloproteinase) in ovulation and tissue remodeling has been suggested. However, the molecular mechanisms regulating key proteinases and identities of essential enzymes that are responsible for the recurring tissue remodeling process have not been established. Our preliminary data suggest that a member of ADAMTS family, ADAMTS9, is likely a downstream target of nuclear progestin receptor (PGR), and is mainly responsible for ovulation. We will use CRISPR/Cas9 to generate global and conditional knockouts of ADAMTS9, and characterize these knockouts to determine whether knockout ADAMTS9 affects ovulation and fertility in zebrafish. If ADAMTS9 is a key downstream enzyme of PGR that is important for ovulation, we should be able to rescue PGR knockout anovulation phenotype by overexpressing ADAMTS9 in PGR knockout fish. In addition, we propose to determine the expression, regulation and functions of ADAMTS9 during ovulation in zebrafish, in order to elucidate the molecular mechanisms and signaling pathways for ovulation. We will determine expression of both transcript and protein of ADAMTS9 during the development of oocytes, and in the follicular cells of preovulatory oocytes around the ovulation in zebrafish. We will also determine whether the expression of ADAMTS9 is hormonally regulated by gonadotropins and/or progestin. We will then determine whether PGR regulates ADAMTS9 directly or indirectly via other transcriptional factors. For the first time, the regulation and functions of ADAMTS9, particularly in the follicular cells of oocytes, will be comprehensively determined in a vertebrate model. The ADAMTS9 knockout models will be very valuable resource for future studies. The functions of ADAMTS9 in ovulation and fertility will also be established in a zebrafish model, which will provide a foundation to study the functions and regulation of ADAMTS9 in mammals, as well as the development of new drug targets and novel non-steroid treatments for infertility, tissue remodeling, and cancer.