Gonadotropin-releasing hormone 3 and gonadotropes relationships in female zebrafish pituitary revealed by single-cell transcriptomics

Gonadotropin-releasing hormone (GnRH) governs reproduction in vertebrates by regulating pituitary gonadotropins. Zebrafish, however, is an exception as gnrh3–/– fish, which lack the hypophysiotropic GnRH3, are fertile, suggesting that zebrafish utilizes a Gnrh-independent mechanism to regulate reproduction. To elucidate the role of Gnrh3 and the Gnrh-independent mechanisms that regulate the pituitary gonadotropes, we profiled the gene expression in individual pituitary cells of wild-type and gnrh–/– adult female zebrafish and identified transcriptionally defined cell types. The classical Lh and Fsh gonadotropes expressed both gonadotropin beta subunits with a ratio of 13:1 (lhb:fshb) and 40:1 (fshb:lhb), respectively. We discovered that Lh gonadotropes predominantly express genes encoding receptors for Gnrh (gnrhr2), thyroid hormone, estrogen, dopamine, and steroidogenic factor 1 (SF1). No Gnrh receptor expression was enriched in Fsh gonadotropes, instead, the expression of cholecystokinin receptor (cckrb) and galanin receptor (gal1rb) were enriched in these cells. The hereditary loss of Gnrh3 gene resulted in downregulation of fshb in Lh gonadotropes. Likewise, targeted chemogenetic ablation of Gnrh3 neurons led to a decrease in the number of fshb+/lhb+ cells. Our studies suggest that Gnrh3 directly acts on Lh gonadotropes through Gnrhr2, but the outcome of this interaction is still unknown. Gnrh3 also regulates fshb expression, probably via a non-Gnrh receptor route. Altogether, while Lh secretion and synthesis are likely regulated by multiple factors in a Gnrh-independent manner, Gnrh3 seems to play a role in the cellular organization of the pituitary in zebrafish. Pituitaries of WT and Gnrh3 knockout female zebrafish were dissociated and analyzed using single cell RNA sequencing