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

In vertebrates, reproduction is 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 to 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 and fertile fshb-/-;lhcgr-/- with LH-Fshr) 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. Males with only fshb or lhb were infertile, with testes dominated by spermatogonia and spermatocytes with few spermatozoa. 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 sex differentiation and gonadal development. This comprehensive genetic study provides insights into functional importance of gonadotropins in zebrafish reproduction and their signaling mechanisms. Author summaryFollicle-stimulating hormone (FSH) and luteinizing hormone (LH) are two key hormones regulating vertebrate reproduction via their specific receptors, FSHR and LHCGR. Zebrafish, like other vertebrates, also has two hormones and two receptors. Interestingly, zebrafish LH signals not only through its cognate receptor Lhcgr but also through FSH receptor Fshr, suggesting existence of three signaling pathways (FSH-Fshr, LH-Fshr and LH-Lhcgr). To investigate the functionality of these pathways, we created various zebrafish mutant lines with all mutational combinations for these hormones and receptors. Our findings demonstrated that Fshr, when activated by either FSH or LH, played a critical role in ovarian growth and folliculogenesis. LH was necessary for oocyte maturation and ovulation, and the LH-Fshr pathway alone was adequate to support the entire process of female reproduction. All three pathways were sufficient to sustain male reproductive function. We further observed that the receptors exhibited low-level spontaneous activities in the absence of hormones, that could support spermatogenesis. Fish lacking all four genes developed only as males with extremely underdeveloped, non-functional testes. This highlights critical roles for gonadotropins not only in fertility, but also in sex differentiation. Our work provides comparative insights into the fundamental mechanisms by which gonadotropins control reproductive processes in vertebrates.