Functional Landscape of Zebrafish Gonadotropins and Receptors: A Comprehensive Genetic Analysis

In vertebrates, reproduction is primarily controlled by two master hormones, or gonadotropins, from the pituitary: follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which act through their cognate receptors, FSHR and LHCGR, in gonads. Like other vertebrates, zebrafish also has two gonadotropins and respective receptors. However, while zebrafish FSH activates Fshr specifically, its LH can activate both Fshr and Lhcgr, suggesting three signaling pathways - the canonical FSH-Fshr and LH-Lhcgr pathways, and a non-canonical LH-Fshr pathway. To dissect functional roles of these pathways, we generated a series of zebrafish mutants lacking one, two, three, or all four genes encoding the two ligands and their receptors: fshb, lhb, fshr, and lhcgr. Single mutants confirmed the essential roles of FSH and LH in zebrafish reproduction. Double mutants demonstrated the functionality of all three pathways, especially the non-canonical LH-Fshr pathway, and highlighted the importance of Fshr in mediating both FSH and LH actions. Transcriptome analysis of double mutant follicles (infertile lhb-/-; lhcgr-/- with FSH-Fshr pathway and fertile fshb-/-; lhcgr-/- with LH-Fshr pathway) revealed potential molecular mechanisms underlying LH stimulation of oocyte maturation and ovulation via Fshr. Triple mutants revealed spontaneous, ligand-independent activities of Fshr and Lhcgr, in supporting spermatogenesis. The quadruple mutant (fshb-/-; lhb-/-; fshr-/-; lhcgr-/-) displayed an all-male phenotype with underdeveloped, infertile gonads primarily containing spermatogonia, suggesting a critical role for gonadotropins in gonadal differentiation and development. This comprehensive genetic study provides insights into functional importance of gonadotropins in zebrafish reproduction and their signaling mechanisms.