This project aims at understanding how organisms cope with a possible loss of essential genes. One of the fundamental biological processes in animals is reproduction that ensures the continuation of a species. Gonadotropin releasing-hormone (GnRH), a protein in the brain, is the governor of reproduction in vertebrates, and its loss of function in many species, including humans, leads to infertility. Surprisingly, zebrafish in which the GnRH gene has been silenced remain fertile. This project examines how reproduction proceeds in zebrafish without GnRH, as well as the possibility that the lack of GnRH triggers yet unknown mechanisms that compensate for its absence so that reproduction, and therefore the species, are maintained. Alternatively, it is possible that zebrafish will prove to be the first known GnRH-independent vertebrate species. Therefore, the study will examine 1) whether GnRH is indispensable for reproduction in zebrafish, 2) if and how a compensation is conveyed, and 3) the role of two potential compensators in replacing GnRH. This study offers an opportunity to reveal a novel adaptation for a critical gene, with the likelihood that this adaptation exists in a broad range of species, and may lead to potential therapies for infertility. The project will support the training of graduate students. Partnering with Carnegie’s BioEYES Baltimore, which provides students in grades 2–12 with hands-on biology experiences using live zebrafish, the project will develop a reproduction-oriented training module for K-12 teachers from Baltimore City middle schools, thereby reaching over 20,000 students.
Normal reproduction in hypothalamic GnRH (GnRH3) knockout (KO) zebrafish can be explained by its dispensability for reproduction or by the activation of a compensatory mechanism. The effect of reproductive impairment induced by 1) focal hypothalamic GnRH3 knockdown via RNAi, in vivo, 2) GnRH receptor blocking by specific antagonists, and 3) Gnrh3 neuron laser-ablation, have indicated that GnRH3 is an active regulator of reproduction in zebrafish. Multiple molecular changes along the reproductive axis of the GnRH3 KO zebrafish, point towards an inherited multi-factorial compensation, ruling out known compensatory mechanisms such as homologous factors (i.e., GnRH2) or neurotransmitters. The hypothesis of the proposal is that GnRH3 is the principal regulator of reproduction in zebrafish and its genetic loss activates inherited complex compensatory machinery along the brain-pituitary axis. The role of GnRH3 in the control of reproduction will be determined via knockdown of Gnrh3 through viral delivery of RNAi, and KO of Gnrh receptors in the brain as well as specifically in Lh and Fsh gonadotropes. The role of GnRH3 neurons (versus GnRH3 peptide) in controlling reproduction will be studied using the GnRH3 KO line and GnRH3 conditional neuronal ablation by cytotoxins. Possible compensating factors that mimic GnRH3 function within GnRH3 neurons will be identified in FACS isolated GnRH3 neurons, followed by Transcriptomics and Proteomics. Two promising non-GnRH3 neuronal neuropeptides that may be replacement candidates for GnRH3 (or that work in conjunction) will be tested through their gene KO and by determining if GnRH3 peptide modulates their expression through histone modifications.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
